Isoxazole beta-lactamase inhibitors

ABSTRACT

β-Lactamase inhibitor compounds (BLIs) are disclosed, including compounds that have activity against class A, class C or class D β-lactamases. Methods of manufacturing the BLIs, and uses of the compounds in the preparation of pharmaceutical compositions and antibacterial applications are also disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/618,127, filed Mar. 30, 2012, and U.S. Provisional Application No.61/790,248, filed Mar. 15, 2013. The entire contents of theseapplications are incorporated herein by reference in their entireties.

TECHNICAL FIELD

This disclosure is directed to β-lactamase inhibitors (BLIs) which areeffective as inhibitors of β-lactamases and, when used in combinationwith β-lactam antibiotics are useful in the treatment of bacterialinfections. The compounds when combined with a β-lactam antibiotic areeffective in treating infections caused by bacteria that are resistantto β-lactam antibiotics due to the presence of β-lactamases.Pharmaceutical compositions comprising such compounds, methods of usingsuch compounds, and processes for preparing such compounds are alsodisclosed.

BACKGROUND

Bacterial resistance to β-lactam antibiotics, especially inGram-negative bacteria, is most commonly mediated by β-lactamases.β-lactamases are enzymes that catalyze the hydrolysis of the β-lactamring, which inactivates the antibacterial activity of the β-lactamantibiotic and allows the bacteria to become resistant. Inhibition ofthe β-lactamase with a BLI slows or prevents degradation of the β-lactamantibiotic and restores β-lactam antibiotic susceptibility toβ-lactamase producing bacteria. Many of these β-lactamases are noteffectively inhibited by BLIs currently on the market rendering theβ-lactam antibiotics ineffective in treating bacteria that produce theseβ-lactamases. There is an urgent need for novel BLIs that inhibitβ-lactamases that are not effectively inhibited by the current clinicalBLIs (e.g. KPC, class C and class Dβ-lactamases) and that could be usedin combination with β-lactam antibiotics to treat infections caused byβ-lactam resistant bacteria.

SUMMARY OF INVENTION

The present invention provides, in one aspect, compounds of chemicalformula (I), or pharmaceutically-acceptable salts thereof, which areBLIs and are useful in combination with β-lactam antibiotics for thetreatment of bacterial infections.

whereinR is selected from

andR¹ is selected from:

a.

-   -   wherein R² is selected from

-   -   wherein each of R³, R⁴ and R⁵ is independently selected from        hydrogen, (C₁-C₃)-alkyl, aminoalkyl, aminocycloalkyl, and        hydroxyalkyl, and n is selected from 1, 2 and 3;

b.

-   -   wherein R⁶ is selected from    -   H and

c.

-   -   wherein R⁷ is selected from H, (C₁-C₃)-unsubstituted alkyl,        amino-(C₂-C₃)-alkyl, amino cyclo alkyl, hydroxyalkyl,    -   and

-   -   and wherein each of p and q is independently selected from 1 and        2.

In another aspect, the invention provides compounds of chemical Formula(A-I) or a pharmaceutically acceptable salt thereof, which are BLIs andare useful in combination with β-lactam antibiotics for the treatment ofbacterial infections.

wherein

R* is selected from

and

R^(1*) is selected from:

a.

-   -   wherein R^(2*) is selected from

-   -   R^(3*) is selected from hydrogen, (C₁-C₃)-alkyl, aminoalkyl,        aminocycloalkyl, hydroxyalkyl,

-   -   each of R^(4*), R^(5*), R^(6*)and R^(7*) is independently        selected from hydrogen, (C₁-C₆)-alkyl, aminoalkyl,        aminocycloalkyl, and hydroxyalkyl, provided that at least one of        R^(4*), R^(5*), R^(6*)and R^(7*)is hydrogen,    -   n is selected from 1, 2, 3 and 4, and    -   m is selected from 1, 2 and 3;

b.

-   -   wherein R⁸ is selected from —NH(C₁-C₃)-alkyl and

-   -   wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as described        previously;

c.

-   -   wherein Z is selected from CR⁹R¹⁰ and NR¹¹,    -   each of R⁹ and R¹⁰ is independently selected from H, NH₂,        —NH(C₁-C₃)— alkyl and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously,

-   -   alternatively, R⁹ and R¹⁰ together with the carbon to which they        are attached, form a cycloalkyl or heterocyclyl ring containing        4-6 ring members,    -   R¹¹ is selected from H and

-   -   each of R¹², R¹³ and R¹⁴ is independently selected from        hydrogen, (C₁-C₆)-alkyl, aminoalkyl, aminocycloalkyl, and        hydroxyalkyl, provided that at least one of R¹², R¹³ and R¹⁴ is        hydrogen,    -   R¹⁵ is selected from NH₂ and

-   -   wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as described        previously,    -   each of p* and q* is independently selected from 0, 1, 2 and 3,    -   T is selected from NH and O,    -   t is selected from 0, 1, 2, 3, and 4, and    -   each of r and y is independently selected from 0 and 1;

d.

-   -   wherein R¹⁶ is selected from NH₂, —NH(C₁-C₃)-alkyl and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*) is as describedpreviously,

-   -   s is selected from 0 and 1, and,    -   v is selected from 0, 1, 2, and 3;

e.

-   -   wherein R¹⁸ is selected from NH₂ and

wherein each

-   -   of R^(4*), R^(5*), R^(6*)and R^(7*) is as described previously,    -   R¹⁷ is selected from amino and hydroxyl, and    -   w is selected from 0 and 1;

f.

g.

-   -   wherein M is selected from NR¹⁹, CR²⁰R²¹ and O,    -   wherein R¹⁹ is selected from H and

where each of R¹²,

-   -   R¹³ and R¹⁴ is as described previously,    -   each of R²⁰ and R²¹ is independently selected from H, NH₂ and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously, and

-   -   u is selected from 0, 1 and 2; and

h.

In one embodiment, the invention provides use of a compound of Formula Ifor inhibiting β-lactamases.

In one embodiment, the invention provides use of a compound of FormulaA-I for inhibiting β-lactamases.

In one embodiment, the invention provides compounds of Formula I withhigh binding affinity for β-lactamase enzymes.

In one embodiment, the invention provides compounds of Formula A-I withhigh binding affinity for β-lactamase enzymes.

In one embodiment, the present invention also provides antibacterialcompositions comprising compounds of Formula I and at least one β-lactamantibiotic.

In one embodiment, the present invention also provides antibacterialcompositions comprising compounds of Formula A-I and at least oneβ-lactam antibiotic.

In one embodiment, the present invention provides pharmaceuticalcompositions comprising compounds of Formula I and at least one β-lactamantibiotic and methods of use thereof.

In one embodiment, the present invention provides pharmaceuticalcompositions comprising compounds of Formula A-I and at least oneβ-lactam antibiotic and methods of use thereof.

In one embodiment, the invention provides methods of use of thecompounds of Formula I to treat bacterial infections in a subject.

In one embodiment, the invention provides methods of use of thecompounds of Formula A-I to treat bacterial infections in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show Table I, Representative Compounds of Formula A-II

FIGS. 2A-2B show Table II, Standard BLI potentiation MIC assay against apanel of isogenic and clinical strains expressing β-lactamases.

FIGS. 3A-3B show Table III, the synergy MIC of representative compoundsof Formula II-A against a panel of isogenic and clinical strainsexpressing β-lactamases.

FIG. 4 shows Table IV, an assay to determine inhibition kinetics ofrepresentative compounds of Formula II-A for the KPC-2 β-lactamase.

FIGS. 5A-5B show Table V, Synergy MIC of Comparator Compounds Against aPanel of Isogenic and Clinical Strains Expressing β-lactamases

DETAILED DESCRIPTION Definitions

Molecular terms, when used in this application, have their commonmeaning unless otherwise specified.

The term “alkyl” is defined as a linear or branched, saturated radicalhaving one to about twenty carbon atoms unless otherwise specified.Preferred alkyl radicals are “lower alkyl” radicals having one to aboutfive carbon atoms. Examples of alkyl groups include, without limitation,methyl, ethyl, tert-butyl, isopropyl, and hexyl. A subset of the termalkyl is “(C₁-C₃)-unsubstituted alkyl” which is defined as an alkylgroup that bears no substituent groups. Examples of(C₁-C₃)-unsubstituted alkyl groups include methyl, ethyl, propyl andisopropyl. It is understood that if a (C₁-C₃)-alkyl is “substituted”that one or more hydrogen atoms is replaced by a substitutent.

The term amino denotes a NH₂ radical.

The term “aminoalkyl” denotes an alkyl in which one or more of the alkylhydrogen atoms has been replaced by an amino group.

The term “aminocycloalkyl” denotes a cycloalkyl in which one of thecycloalkyl hydrogen atoms has been replaced by an amino group.

The term “cycloalkyl” or “cycloalkyl ring” is defined as a saturated orpartially unsaturated carbocyclic ring in a single or fused carbocyclicring system having from three to twelve ring members. In a preferredembodiment, a cycloalkyl is a ring system having three to seven ringmembers. Examples of a cycloalkyl group include, without limitation,cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.

The term “hydroxyalkyl” denotes an alkyl radical in which one or more ofthe alkyl hydrogen atoms has been replaced by a hydroxyl group.

It will be understood by one of skill in the art that a

or denote the point of attachment of a substituent group whereindicated. For example

or —C(O)NHR⁵ represent that the point of attachment of the amide moietyis at the carbonyl carbon.

The functional classification of β-lactamases and terms “Class A”,“Class C”, and “Class D” β-lactamases are understood by one of skill inthe art and are described in “Updated Functional Classification ofβ-Lactamases”, Bush, K.; Jacoby, G. A.; Antimicrob. Agents Chemother.2010, 54, 969-976, herein incorporated by reference.

The salts of the compounds of the invention include acid addition saltsand base addition salts. In a one embodiment, the salt is apharmaceutically acceptable salt of the compound of Formula I. The term“pharmaceutically acceptable salts” embraces salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. The nature of the salt is not critical, provided that it ispharmaceutically-acceptable. Suitable pharmaceutically acceptable acidaddition salts of the compounds of the invention may be prepared from aninorganic acid or an organic acid. Examples of such inorganic acidsinclude, without limitation, hydrochloric, hydrobromic, hydroiodic,nitric, carbonic, sulfuric and phosphoric acid. Examples of appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which include, without limitation, formic, acetic,propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic),methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic,benzenesulfonic, toluenesulfonic, sulfanilic, mesylic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic,galactic, and galacturonic acid. Suitable pharmaceutically-acceptablebase addition salts of compounds of the invention include, but are notlimited to, metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, lysine and procaine. All of thesesalts may be prepared by conventional means from the correspondingcompound of the invention by treating, for example, the compound of theinvention with the appropriate acid or base.

The compounds of the invention can possess one or more asymmetric carbonatoms and are thus capable of existing in the form of optical isomers aswell as in the form of racemic or non-racemic mixtures thereof. Thecompounds of the invention can be utilized in the present invention as asingle isomer or as a mixture of stereochemical isomeric forms.Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can beseparated by conventional means such as chromatography, distillation,crystallization or sublimation. The optical isomers can be obtained byresolution of the racemic mixtures according to conventional processes,for example by formation of diastereoisomeric salts by treatment with anoptically active acid or base. Examples of appropriate acids include,without limitation, tartaric, diacetyltartaric, dibenzoyltartaric,ditoluoyltartaric and camphorsulfonic acid. The mixture of diastereomerscan be separated by crystallization followed by liberation of theoptically active bases from the optically active salts. An alternativeprocess for separation of optical isomers includes the use of a chiralchromatography column optimally chosen to maximize the separation of theenantiomers. Still another available method involves synthesis ofcovalent diastereoisomeric molecules by treating compounds of theinvention with an optically pure acid in an activated form or anoptically pure isocyanate. The synthesized diastereoisomers can beseparated by conventional means such as chromatography, distillation,crystallization or sublimation, and then hydrolyzed to obtain theenantiomerically pure compound. The optically active compounds of theinvention can likewise be obtained by utilizing optically activestarting materials. These isomers may be in the form of a free acid, afree base, an ester or a salt.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In another embodiment, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet anotherembodiment, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

The invention also embraces isolated compounds. An isolated compoundrefers to a compound which represents at least 10%, such as at least20%, such as at least 50% and further such as at least 80% of thecompound present in the mixture. In one embodiment, the compound, apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising the compound exhibits detectable (i.e.statistically significant) activity when tested in conventionalbiological assays such as those described herein.

β-Lactamase Inhibitors (BLIs)

In one aspect, the invention provides compounds of Formula I orpharmaceutically-acceptable salts thereof:

Substituent R of Formula I is selected from

In a preferred embodiment, R is

The group R¹ of Formula I is selected from:

a.

-   -   wherein R² is selected from

-   -   wherein each of R³, R⁴ and R⁵ is independently selected from        hydrogen, (C₁-C₃)-alkyl, aminoalkyl, aminocycloalkyl, and        hydroxyalkyl, and n is selected from 1, 2 and 3;

b.

-   -   wherein R⁶ is selected from    -   H and

c.

-   -   wherein R⁷ is selected from H, (C₁-C₃)-unsubstituted alkyl,        amino-(C₂-C₃)-alkyl, amino cyclo alkyl, hydroxyalkyl,    -   and

-   -   and wherein each of p and q is independently selected from 1 and        2.

In one aspect of the invention n is 1. In another aspect of theinvention n is 2. In another aspect of the invention n is 3.

In one aspect R¹ is selected from —CH₂NH₂, —CH₂CH₂NH₂, —CONH(CH₂)₂NH₂,

In one embodiment of the invention, R¹ is selected from

In one embodiment of the invention, the compounds of the invention areof the stereochemistry disclosed in Formula II.

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is —CH₂NH₂.

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is —CH₂CH₂NH₂.

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is —CONH(CH₂)₂NH₂.

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is

In another embodiment of the invention, the compound is of Formula IIand R is —OSO₃H and R¹ is

Preferred compounds of Formula I are the compounds:

It will be understood by one of skill in the art that depending on thenature of R¹ and R, compounds of Formula I may exist in a salt orzwitterionic form.

In one aspect, the invention provides compounds of Formula A-I orpharmaceutically-acceptable salts thereof:

Substituent R* of Formula A-I is selected from

In a preferred embodiment, R^(*) is

The group R^(1*) is selected from:

a.

-   -   wherein R^(2*) is selected from

-   -   R^(3*) is selected from hydrogen, (C₁-C₃)-alkyl, aminoalkyl,        aminocycloalkyl, hydroxyalkyl,

-   -   each of R^(4*), R^(5*), R^(6*)and R^(7*) is independently        selected from hydrogen, (C₁-C₆)-alkyl, aminoalkyl,        aminocycloalkyl, and hydroxyalkyl, provided that at least one of        R^(4*), R^(5*), R^(6*)and R^(7*)is hydrogen,    -   n is selected from 1, 2, 3 and 4, and    -   m is selected from 1, 2 and 3;

b.

-   -   wherein R⁸ is selected from —NH(C₁-C₃)-alkyl and

-   -   wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as described        previously;

c.

-   -   wherein Z is selected from CR⁹R¹⁰ and NR¹¹,    -   each of R⁹ and R¹⁰ is independently selected from H, NH₂,        —NH(C₁-C₃)—alkyl and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously,

-   -   alternatively, R⁹ and R¹⁰ together with the carbon to which they        are attached, form a cycloalkyl or heterocyclyl ring containing        4-6 ring members,    -   R¹¹ is selected from H and

-   -   each of R¹², R¹³ and R¹⁴ is independently selected from        hydrogen, (C₁-C₆)-alkyl, aminoalkyl, aminocycloalkyl, and        hydroxyalkyl, provided that at least one of R¹², R¹³ and R¹⁴ is        hydrogen,    -   R¹⁵ is selected from NH₂ and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously,

-   -   each of p* and q* is independently selected from 0, 1, 2 and 3,    -   T is selected from NH and O,    -   t is selected from 0, 1, 2, 3, and 4, and    -   each of r and y is independently selected from 0 and 1;

d.

-   -   wherein R¹⁶ is selected from NH₂, —NH(C₁-C₃)-alkyl and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*) is as describedpreviously,

-   -   s is selected from 0 and 1, and,    -   v is selected from 0, 1, 2, and 3;

e.

-   -   wherein R¹⁸ is selected from NH₂ and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*) is as describedpreviously,

-   -   R¹⁷ is selected from amino and hydroxyl, and    -   w is selected from 0 and 1;

f.

g.

-   -   wherein M is selected from NR¹⁹, CR²⁰R²¹ and O,    -   wherein R¹⁹ is selected from H and

where each of R¹², R¹³ and R¹⁴ is as described previously,

-   -   each of R²⁰ and R²¹ is independently selected from H, NH₂ and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously, and

-   -   u is selected from 0, 1 and 2; and

h.

In one aspect of the invention R^(1*) is selected from

In one embodiment of the invention R^(1*) is selected from

In one embodiment of the invention, the compounds of the invention areof the stereochemistry disclosed in Formula A-II.

In another embodiment of the invention, R^(*) and R^(1*) are chosen fromthe substituents listed in Table I (See Figure I). Preferred compoundsof Formula A-I are

It will be understood by one of skill in the art that depending on thenature of R^(1*) and R*, compounds of Formula I may exist in a salt orzwitterionic form.

Enzyme Inhibition and Binding Affinity

The compounds of the invention (e.g. compounds of Formula I, compoundsof Formula A-I, compounds of Formula II, compounds of Formula A-II) areeffective in inhibiting β-lactamase. In one aspect of the invention thecompounds of Table I are effective β-lactamase inhibitors. In one aspectthe compound

is effective in inhibiting β-lactamase. In one aspect the compound

is effective in inhibiting β-lactamase

When used in combination with β-lactam antibiotics, the compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) potentiate theactivity of the β-lactam antibiotic against microorganisms that arenormally resistant to β-lactam antibiotics due to the presence of aβ-lactamase or multiple β-lactamases.

In one aspect of the invention the compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) inhibit β-lactamases selected from classA, class C or class D β-lactamases. In one aspect of the invention thecompounds of Formula I, inhibit β-lactamases selected from class A,class C or class D β-lactamases. In one aspect of the invention thecompounds of Formula A-I inhibit β-lactamases selected from class A,class C or class D β-lactamases. In one aspect of the invention thecompounds of Formula II inhibit β-lactamases selected from class A,class C or class D β-lactamases. In one aspect of the invention thecompounds of Formula A-II inhibit β-lactamases selected from class A,class C or class Dβ-lactamases. In one aspect of the invention thecompounds of Table I inhibit β-lactamases selected from class A, class Cor class D β-lactamases. In one aspect of the invention the compound ofFormula

inhibits β-lactamases selected from class A, class C or classDβ-lactamases. In one aspect of the invention the compound of Formula

inhibits β-lactamases selected from class A, class C or classDβ-lactamases. Class A β-lactamases for example, include, but are notlimited to, TEM, SHV, CTX-M, KPC, GES, VEB, SME, and GEX. In a preferredaspect of the invention, the compounds of the invention (e.g. compoundsof Formula I, compounds of Formula A-I, compounds of Formula II,compounds of Formula A-II) inhibit KPC β-lactamases. In a preferredaspect of the invention, the compounds of Formula I inhibit KPCβ-lactamases. In a preferred aspect of the invention, the compounds ofFormula A-I inhibit KPC β-lactamases. In a preferred aspect of theinvention, the compounds of Formula II inhibit KPC β-lactamases. In apreferred aspect of the invention, the compounds of Formula A-II inhibitKPC β-lactamases. More preferably the compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) inhibit KPC-2 or KPC-3 β-lactamases. Morepreferably the compounds of Formula I inhibit KPC-2 or KPC-3β-lactamases. More preferably the compounds of Formula A-I inhibit KPC-2or KPC-3 β-lactamases. More preferably the compounds of Formula IIinhibit KPC-2 or KPC-3 β-lactamases. More preferably the compounds ofFormula A-II inhibit KPC-2 or KPC-3 β-lactamases. In one aspect of theinvention, the compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) inhibit KPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2,Table II). In one aspect of the invention, the compounds of Formula Iinhibit KPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2, TableII). In one aspect of the invention, the compounds of Formula A-Iinhibit KPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2, TableII). In one aspect of the invention, the compounds of Formula II inhibitKPC-2 or KPC-3 β-lactamases in clinical strains (FIG. 2, Table II). Inone aspect of the invention, the compounds of Formula A-II inhibit KPC-2or KPC-3 β-lactamases in clinical strains (FIG. 2, Table II). Class Cβ-lactamases for example, include, but are not limited to chromosomalAmpCs, and plasmid based ACC, DHA, CMY, FOX, ACT, MIR, LAT, MOXβ-lactamases. Class D β-lactamase enzymes, for example, include, but arenot limited to oxacillinases or OXA β-lactamases. In a preferred aspectof the invention, the compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) inhibit OXA-15 β-lactamases. In a preferred aspect ofthe invention, the compounds of Formula I inhibit OXA-15 β-lactamases.In a preferred aspect of the invention, the compounds of Formula A-Iinhibit OXA-15 β-lactamases. In a preferred aspect of the invention, thecompounds of Formula II inhibit OXA-15 β-lactamases. In a preferredaspect of the invention, the compounds of Formula A-II inhibit OXA-15β-lactamases.

Unless otherwise indicated, the activity of the BLI compounds can bedescribed by the MIC value obtained from a Synergy MIC assay or a BLIpotentiation assay (e.g as described herein), both of which are run inthe presence of a β-lactam. The lower the sMIC or MIC value the moreactive the BLI, regardless of the mechanism of action of the BLIcompound (e.g., including inhibition of β-lactamases by the BLI or anyother mechanism of action or combination of mechanisms of action). ThesMIC and BLI potentiation assay data supports that the compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) potentiate (i.e.make more potent) the activity of the β-lactam antibiotic againstβ-lactamase producing strains by inhibiting the β-lactamase.

In one embodiment, the BLI activity is measured by growth inhibition ofa β-lactamase producing bacterial strains in a Synergy MIC (sMIC) assay.Preferably, the sMIC value for the compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) is 8 μg/mL or less. In a more preferredaspect of the invention, the sMIC value for the compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) is 4 μg/mL to 8μg/mL. In an even more preferred aspect of the invention, the sMIC valuefor the compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) is 1 to 2 μg/mL. In a still more preferred aspect of theinvention, the sMIC value for the compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) is 0.2 to 0.5 μg/mL. Synergy MICs forrepresentative compounds of the invention are described in Table III(See FIG. 3). It will be understood by one of skill in the art that thegrowth inhibition of β-lactamase producing strains can also be measuredby a checkerboard synergy assay like that disclosed in InternationalPatent Application Number WO 2008/039420 or a standard BLI potentiationassay using a fixed concentration of BLI.

In one embodiment, the BLI activity is measured by growth inhibition ofa β-lactamase producing bacterial strains in a standard BLI potentiationassay using a fixed concentration of BLI. Preferably, the MIC value forthe compounds of the invention (e.g. compounds of Formula I, compoundsof Formula A-I, compounds of Formula II, compounds of Formula A-II) is 8μg/mL or less. In a more preferred aspect of the invention, the MICvalue for the compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) is 4 to 8 μg/mL. In an even more preferred aspect of theinvention, the MIC value for the compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) is 1 to 2 μg/mL. In a still morepreferred aspect of the invention, the MIC value for the compounds ofthe invention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) is 0.2 μg/mL to 0.5μg/mL.

The compounds of the present invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) have a broad spectrum of activity across a wide variety ofβ-lactamase producing bacteria. It was surprisingly found that thecompounds of the present invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) are active in potentiating activity of β-lactam antibiotics, inparticular, Ceftolozane, against strains expressing class D β-lactamaseOXA-15 β-lactamase. Currently marketed BLIs inhibit most of the class Aβ-lactamases, but poorly inhibit class A KPC β-lactamases and class Cβ-lactamases and have variable success in inhibiting penicillinase andcarbapenemase-type class D β-lactamases. The compounds of the presentinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) are active against awide variety of bacterial strains that express class A and Cβ-lactamases and also, surprisingly are active against bacterial strainsthat express the class D cephalosporinase OXA-15 (Tables II and III).This increased activity against the class D β-lactamase is criticalbecause differential effectiveness against different types ofβ-lactamase producing bacteria is necessary in order to effectively useβ-lactam antibiotics to treat resistant strains of bacteria (videinfra).

In one embodiment, the compounds the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) are unexpectedly more active against bacterial strainsthat express OXA-15 β-lactamases than the most structurally similarcompound, Avibactam (comparator compound CCC). Compounds that are moreactive than Avibactam against bacterial strains that express the class Dcephalosporinase OXA-15 are, for example, compounds 603, 604, 611, 614,618.

In one embodiment, the compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) are unexpectedly more active against and/or showbroader spectrum of activity against bacterial strains that express KPCβ-lactamases than the most structurally similar compound, Avibactam.Compounds that are more active than Avibactam for at least one,bacterial strain that expresses KPC β-lactamase and/or show a betterspectrum of activity against bacterial strains that express KPCβ-lactamases than Avibactam are, for example, compounds 601, 602, 603,604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617,618, 620, 621, 622, 623, 624, 625, 626, and 627.

In another aspect of the invention, the compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) have high binding affinity for theβ-lactamase enzyme. Consequently these compounds are better inhibitorsof the β-lactamase enzyme. The inhibition kinetics of the compounds ofthe invention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) was measuredaccording to the procedure outlined in Example 37. The compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) have a high bindingaffinity for the β-lactamase enzyme.

In one embodiment the compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) have a binding affinity of 1000-5000 mM⁻¹s⁻¹. Compoundsthat have a binding affinity of 1000-5000 mM⁻¹s⁻¹ are, for example,compound 604 and 608 (Table IV).

In one embodiment the compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) have a binding affinity of 100-999 mM⁻¹s⁻¹. Compoundsthat have a binding affinity of 100-999 mM⁻¹s⁻¹ are, for example,compounds 601, 603, 605, 606, 607, 609, 610, 611, 612, 613, 614, 615,616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, and 627 (TableIV).

In one embodiment the compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) have a binding affinity of 1-99 mM⁻¹s⁻¹. Compounds thathave a binding affinity of 1-99 mM⁻¹s⁻¹ are, for example, 602 (TableIV).

It was surprisingly found that the compounds of the present inventionhave a higher binding affinity for the β-lactamase enzyme than theclosest structural comparator Avibactam (Table IV, See FIG. 4).

The compounds of the invention were also shown to be better BLIs thanother comparator compounds as shown in FIG. 5.

Pharmaceutical Compositions Comprising the Compounds of the Inventionand Use Thereof

Another object of the invention is pharmaceutical compositions orformulations comprising compounds the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II), or salts thereof, preferably further comprising aβ-lactam antibiotic. In one embodiment of the invention ispharmaceutical compositions or formulations comprising compounds ofFormula I, or salts thereof, preferably further comprising a β-lactamantibiotic. In one embodiment of the invention is pharmaceuticalcompositions or formulations comprising compounds of Formula A-I, orsalts thereof, preferably further comprising a β-lactam antibiotic. Inone embodiment of the invention is pharmaceutical compositions orformulations comprising compounds of Formula II, or salts thereof,preferably further comprising a β-lactam antibiotic. In one embodimentof the invention is pharmaceutical compositions or formulationscomprising compounds of Formula A-II, or salts thereof, preferablyfurther comprising a β-lactam antibiotic. In one embodiment of theinvention is pharmaceutical compositions or formulations comprisingcompounds of Table I. In one embodiment of the invention ispharmaceutical compositions or formulations comprising compounds ofFormula

or salts thereof, preferably further comprising a β-lactam antibiotic.In one embodiment of the invention is pharmaceutical compositions orformulations comprising compounds of Formula

or salts thereof, preferably further comprising a β-lactam antibiotic.

The pharmaceutical compositions can be formulated for oral, intravenous,intramuscular, subcutaneous or parenteral administration for thetherapeutic or prophylactic treatment of diseases, such as bacterialinfections. Preferably, the pharmaceutical composition is formulated forintravenous administration.

The pharmaceutical preparations disclosed herein may be prepared inaccordance with standard procedures and are administered at dosages thatare selected to reduce, prevent or eliminate infection (see, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa. and Goodman and Gilman's “The Pharmaceutical Basis of Therapeutics,”Pergamon Press, New York, N.Y., the contents of which are incorporatedherein by reference, for a general description of the methods foradministering various antimicrobial agents for human therapy).

The pharmaceutical compositions can comprise one or more of thecompounds disclosed herein (e.g. one or more compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably a compound of Formula A-I or Formula A-II, inconjunction with a β-lactam antibiotic, in association with one or morenontoxic, pharmaceutically-acceptable carriers and/or diluents and/oradjuvants and/or excipients. As used herein, the phrase“pharmaceutically-acceptable carrier” refers to any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, that arecompatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Non-limiting examples of carriers and excipients include corn starch orgelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol,dicalcium phosphate, sodium chloride and alginic acid. The compositionsmay contain croscarmellose sodium, microcrystalline cellulose, cornstarch, sodium starch glycolate and alginic acid.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metallicstearates, stearic acid, silicone fluid, talc, waxes, oils and colloidalsilica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. It may also be desirable to adda coloring agent to make the dosage form more aesthetic in appearance orto help identify the product.

For oral or parenteral administration, compounds of the presentinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) preferably acompound of Formula A-I or Formula A-II, in conjunction with a β-lactamantibiotic, can be mixed with conventional pharmaceutical carriers andexcipients and used in the form of tablets, capsules, elixirs,suspensions, syrups, wafers and the like. The compositions comprising acompound of this invention may contain from about 0.1% to about 99% byweight of the active compound, such as from about 10% to about 30%.

For oral use, solid formulations such as tablets and capsules areuseful. Sustained release or enterically coated preparations may also bedevised. For pediatric and geriatric applications, one embodimentprovides suspensions, syrups and chewable tablets. For oraladministration, the pharmaceutical compositions are in the form of, forexample, a tablet, capsule, suspension or liquid.

The pharmaceutical compositions may be made in the form of a dosage unitcontaining a therapeutically-effective amount of the active ingredient.Examples of such dosage units are tablets and capsules. For therapeuticpurposes, the tablets and capsules which can contain, in addition to theactive ingredient, conventional carriers such as binding agents, forexample, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, ortragacanth; fillers, for example, calcium phosphate, glycine, lactose,maize-starch, sorbitol, or sucrose; lubricants, for example, magnesiumstearate, polyethylene glycol, silica, or talc; disintegrants, forexample, potato starch, flavoring or coloring agents, or acceptablewetting agents. Oral liquid preparations generally are in the form ofaqueous or oily solutions, suspensions, emulsions, syrups or elixirs,preparations of the invention may contain conventional additives such assuspending agents, emulsifying agents, non-aqueous agents,preservatives, coloring agents and flavoring agents. Non-limitingexamples of additives for liquid preparations include acacia, almondoil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup,glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methylor propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbicacid.

For intravenous (IV) use, the pharmaceutical composition (e.g. compoundsof Formula I, compounds of Formula A-I, compounds of Formula II,compounds of Formula A-II) preferably a compound of Formula A-I orFormula A-II, in conjunction with a β-lactam antibiotic, can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion. Intravenous fluids include, withoutlimitation, physiological saline or Ringer's solution. Intravenousadministration may be accomplished by using, without limitation,syringe, mini-pump or intravenous line.

Pharmaceutical compositions of this invention (e.g. compounds of FormulaI, compounds of Formula A-I, compounds of Formula II, compounds ofFormula A-II) preferably a compound of Formula A-I or Formula A-II, forparenteral injection comprise pharmaceutically-acceptable aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnon-aqueous carriers, diluents, solvents or vehicles include water,ethanol, benzyl alcohol, polyols (such as glycerol, propylene glycol,and polyethylene glycol), and suitable mixtures thereof, vegetable oils(such as corn oil or olive oil), and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by the useof coating materials such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants. The compositions can include various buffers.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. They may alsocontain taggants or other anti-counterfeiting agents, which are wellknown in the art. Prevention of the action of microorganisms may beensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, and phenol sorbic acid. It may alsobe desirable to include isotonic agents such as sugars and sodiumchloride. Prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption, suchas aluminum monostearate and gelatin.

Injectable depot forms can be made by forming microencapsulatingmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations can also be prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions, which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. Such forms may include forms that dissolveor disintegrate quickly in the oral environment. In such solid dosageforms, the active compound preferably a compound of Formula A-I orFormula A-II in conjunction with a β-lactam antibiotic, can be mixedwith at least one inert, pharmaceutically-acceptable excipient orcarrier. Suitable excipients include, for example, (a) fillers orextenders such as starches, lactose, sucrose, glucose, mannitol, andsilicic acid; (b) binders such as cellulose and cellulose derivatives(such as hydroxypropylmethylcellulose, hydroxypropylcellulose, andcarboxymethylcellulose), alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia; (c) humectants such as glycerol; (d) disintegratingagents such as sodium starch glycolate, croscarmellose, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate; (e) solution retarding agents such asparaffin; (f) absorption accelerators such as quaternary ammoniumcompounds; (g) wetting agents, such as cetyl alcohol and glycerolmonostearate, fatty acid esters of sorbitan, poloxamers, andpolyethylene glycols; (h) absorbents such as kaolin and bentonite clay;(i) lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(j) glidants such as talc, and silicone dioxide. Other suitableexcipients include, for example, sodium citrate or dicalcium phosphate.The dosage forms may also comprise buffering agents.

Solid dosage forms, including those of tablets, dragees, capsules,pills, and granules, can be prepared with coatings and shells such asfunctional and aesthetic enteric coatings and other coatings well knownin the pharmaceutical formulating art. They may optionally containopacifying agents and colorants. They may also be in a form capable ofcontrolled or sustained release. Examples of embedding compositions thatcan be used for such purposes include polymeric substances and waxes.

The pharmaceutical compositions can be delivered using controlled (e.g.,capsules) or sustained release (e.g., bioerodable matrices) deliverysystems. Exemplary delayed release delivery systems for drug deliverythat are suitable for administering the pharmaceutical compositions aredescribed in U.S. Pat. No. 4,452,775 (issued to Kent), U.S. Pat. No.5,039,660 (issued to Leonard), and U.S. Pat. No. 3,854,480 (issued toZaffaroni).

In some cases, in order to prolong the effect of the drug, it may bedesirable to slow the absorption of the drug following subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. Amorphous material may be used alone or together withstabilizers as necessary. The rate of absorption of the drug thendepends upon its rate of dissolution, which in turn, may depend uponcrystal size and crystalline form.

Alternatively, delayed absorption of a parenterally administered drugform can be accomplished by dissolving or suspending the drug in an oilvehicle.

For intramuscular preparations, a sterile formulation of compounds,preferably a compound of Formula A-I or Formula A-II in conjunction witha β-lactam antibiotic, or suitable soluble salt forms thereof, forexample hydrochloride salts, can be dissolved and administered in apharmaceutical diluent such as Water-for-Injection (WFI), physiologicalsaline or 5% glucose. A suitable insoluble form of the compound may beprepared and administered as a suspension in an aqueous base or apharmaceutically acceptable oil base, e.g., an ester of a long chainfatty acid such as ethyl oleate.

A dose of an intravenous, intramuscular, or parental formulation ofcompounds, preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, may be administered as a bolusor by slow infusion. A bolus is a dose that is administered in less than30 minutes. In one embodiment, a bolus is administered in less than 15or less than 10 minutes. In another embodiment, a bolus is administeredin less than 5 minutes. In yet another embodiment, a bolus isadministered in one minute or less. An infusion is a dose that isadministered at a rate of 30 minutes or greater. In one embodiment, theinfusion is one hour or greater. In another embodiment, the infusion issubstantially constant.

For topical use the pharmaceutical compositions, preferably a compoundof Formula A-I or Formula A-II in conjunction with a β-lactamantibiotic, can also be prepared in suitable forms to be applied to theskin, or mucus membranes of the nose and throat, and can take the formof creams, ointments, liquid sprays or inhalants, lozenges, or throatpaints. Such topical formulations further can include chemical compoundssuch as dimethylsulfoxide (DMSO) to facilitate surface penetration ofthe active ingredient.

For application to the eyes or ears, the pharmaceutical composition canbe presented in liquid or semi-liquid form formulated in hydrophobic orhydrophilic bases as ointments, creams, lotions, paints or powders.

For rectal administration, the pharmaceutical compositions, preferably acompound of Formula A-I or Formula A-II in conjunction with a β-lactamantibiotic, can be administered in the form of suppositories admixedwith conventional carriers such as cocoa butter, polyethylene glycol ora suppository wax or other glyceride that are solid at room temperaturebut liquid at body temperature and therefore melt in the rectum orvaginal cavity and release the active compound.

Alternatively, the pharmaceutical compositions can be in powder form forreconstitution in the appropriate pharmaceutically acceptable carrier atthe time of delivery. In another embodiment, the unit dosage form ofcompounds, preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, can be a solution of one or morecompounds, or salts thereof, in a suitable diluent, in sterilehermetically sealed ampoules or sterile syringes. The concentration ofthe compounds, preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, in the unit dosage may vary,e.g. from about 1 percent to about 50 percent, depending on the compoundused and its solubility and the dose desired by the physician. If thecompositions contain dosage units, each dosage unit can contain from1-500 mg of the active material. For adult human treatment, the dosageemployed can range from 5 mg to 10 g, per day, depending on the routeand frequency of administration.

The pharmaceutical compositions disclosed herein can be placed in apharmaceutically acceptable carrier and are delivered to a recipientsubject (e.g., a human) in accordance with known methods of drugdelivery. In general, the methods of delivering the pharmaceuticalcompositions in vivo utilize art-recognized protocols for delivering theagent with the only substantial procedural modification being thesubstitution of the compounds of the present invention for the drugs inthe art-recognized protocols. Likewise, methods for using the claimedcompositions for treating cells in culture, for example, to eliminate orreduce the level of bacterial contamination of a cell culture, utilizeart-recognized protocols for treating cell cultures with antibacterialagent(s) with the only substantial procedural modification being thesubstitution of the compounds of the present invention, preferably incombination with a β-lactam antibiotic for the drugs in theart-recognized protocols.

Exemplary procedures for delivering an antibacterial agent are describedin U.S. Pat. Nos. 6,468,967; 6,852,689; and 5,041,567, issued to Rogersand in PCT patent application number EP94/02552 (publication no. WO95/05384), the disclosures of which are incorporated herein by referencein their entirety. In one embodiment, one or more compounds of theinvention, preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, or pharmaceutical compositionsthereof are administered orally, rectally or via injection (intravenous,intramuscular or subcutaneous). In another embodiment, one or morecompounds of the invention, preferably a compound of Formula A-I orFormula A-II in conjunction with a β-lactam antibiotic, orpharmaceutical compositions thereof are administered orally, rectally orvia injection (intravenous, intramuscular or subcutaneous) to treat aninfection caused by β-lactam resistant bacteria. In another embodiment,one or more compounds of the invention, preferably a compound of FormulaA-I or Formula A-II in conjunction with a β-lactam antibiotic, orpharmaceutical compositions thereof are administered orally to treat aninfection caused by β-lactamase producing bacteria.

As used herein, the phrases “therapeutically-effective dose” and“therapeutically-effective amount” refer to an amount of a compound thatprevents the onset, alleviates the symptoms, stops the progression of abacterial infection, or results in another desired biological outcomesuch as, e.g., improved clinical signs or reduced/elevated levels oflymphocytes and/or antibodies.

The term “treating” or “treatment” is defined as administering, to asubject, a therapeutically-effective amount of one or more compoundsboth to prevent the occurrence of an infection and to control oreliminate an infection. Those in need of treatment may includeindividuals already having a particular medical disease as well as thoseat risk for the disease (i.e., those who are likely to ultimatelyacquire the disorder).

The term “subject,” as used herein, refers to a mammal, a plant, a loweranimal, or a cell culture. In one embodiment, a subject is a human orother animal patient in need of antibacterial treatment.

The term “administering” or “administration” and the like, refers toproviding the compound of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) to the subject in need of treatment. Preferably the subject is amammal, more preferably a human. The present invention comprisesadministering the compound of the invention (e.g. compounds of FormulaI, compounds of Formula A-I, compounds of Formula II, compounds ofFormula A-II) in conjunction with a β-lactam antiobiotic. When acompound of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II) isadministered in conjunction with a β-lactam antiobiotic, the compound ofthe invention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) and the β-lactamantiobiotic can be administered at the same time or different times.When the compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) and the β-lactam antiobiotic are administered at the same time,they can be administered as a single composition or pharmaceuticalcomposition or they can be administered separately. It is understoodthat when a compound of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) is administered in conjunction with a β-lactam antibiotic, thatthe active agents can be administered in a single combination or inmultiple combinations. For example, when administered by IV, thecompound of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II) can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion, then a β-lactam antibiotic can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion. Conversely the β-lactam antibiotic can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion, then a compound of Formula I can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion. Alternatively, a pharmaceuticalcomposition comprising a compound of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) and a β-lactam antibiotic can be dissolved or suspendedin any of the commonly used intravenous fluids and administered byinfusion.

In one embodiment of the invention, is provided a method of treating orpreventing a bacterial infection comprising administering to a subjectin need thereof a therapeutically-effective amount of the pharmaceuticalcomposition comprising a compound of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) and a β-lactam antibiotic. In one embodiment of theinvention, is provided a method of treating or preventing a bacterialinfection comprising administering to a subject in need thereof atherapeutically-effective amount of the pharmaceutical compositioncomprising a compound of Formula I, and a β-lactam antibiotic. In oneembodiment of the invention, is provided a method of treating orpreventing a bacterial infection comprising administering to a subjectin need thereof a therapeutically-effective amount of the pharmaceuticalcomposition comprising a compound of Formula A-I, and a β-lactamantibiotic. In one embodiment of the invention, is provided a method oftreating or preventing a bacterial infection comprising administering toa subject in need thereof a therapeutically-effective amount of thepharmaceutical composition comprising a compound of Formula II, and aβ-lactam antibiotic. In one embodiment of the invention, is provided amethod of treating or preventing a bacterial infection comprisingadministering to a subject in need thereof a therapeutically-effectiveamount of the pharmaceutical composition comprising a compound ofFormula A-II, and a β-lactam antibiotic.

In one embodiment of the invention, is provided a method of treating orpreventing a bacterial infection comprising administering to a subjectin need thereof, a therapeutically-effective amount of a β-lactamantibiotic in conjunction with a compound of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II). In one embodiment of the invention, isprovided a method of treating or preventing a bacterial infectioncomprising administering to a subject in need thereof, atherapeutically-effective amount of a β-lactam antibiotic in conjunctionwith a compound of Formula I. In one embodiment of the invention, isprovided a method of treating or preventing a bacterial infectioncomprising administering to a subject in need thereof, atherapeutically-effective amount of a β-lactam antibiotic in conjunctionwith a compound of Formula A-I. In one embodiment of the invention, isprovided a method of treating or preventing a bacterial infectioncomprising administering to a subject in need thereof, atherapeutically-effective amount of a β-lactam antibiotic in conjunctionwith a compound of Formula II. In one embodiment of the invention, isprovided a method of treating or preventing a bacterial infectioncomprising administering to a subject in need thereof, atherapeutically-effective amount of a β-lactam antibiotic in conjunctionwith a compound of Formula A-II. In one embodiment of the invention, isprovided a method of treating or preventing a bacterial infectioncomprising administering to a subject in need thereof, atherapeutically-effective amount of a β-lactam antibiotic in conjunctionwith a compound of Table I. In one embodiment of the invention, isprovided a method of treating or preventing a bacterial infectioncomprising administering to a subject in need thereof, atherapeutically-effective amount of a β-lactam antibiotic in conjunctionwith a compound of Formula

In one embodiment of the invention, is provided a method of treating orpreventing a bacterial infection comprising administering to a subjectin need thereof, a therapeutically-effective amount of a β-lactamantibiotic in conjunction with a compound of Formula

In one embodiment of the invention, is provided a method of treating orpreventing a bacterial infection in a subject comprising the steps of

-   -   a. administering to the subject a compound of the invention; and    -   b. administering to the subject a therapeutically-effective        amount of a β-lactam antibiotic.

In one embodiment the compound in step a is a compound of Formula I. Inone embodiment the compound in step a is a compound of Formula A-I. Inone embodiment the compound in step a is a compound of Formula II. Inone embodiment the compound in step a is a compound of Formula A-II. Inone embodiment the compound in step a is a compound of Table I. In oneembodiment the compound in step a is a compound of Formula A-II. In oneembodiment the compound in step a is a compound of Formula

In one embodiment the compound in step a is a compound of Formula

In one embodiment, the β-lactam antibiotic in step b is Ceftolozane orCeftazidime. In one embodiment the compound in step a is a compound ofFormula

and the β-lactam antibiotic in step b is Ceftolozane. In one embodimentthe compound in step a is a compound of Formula

and the β-lactam antibiotic in step b is Ceftolozane.

In one embodiment of the invention, is provided a method of treating orpreventing a bacterial infection in a subject comprising the steps of

-   -   a. administering to the subject a therapeutically-effective        amount of a β-lactam antibiotic; and    -   b. administering to the subject a compound of the invention.

In one embodiment the compound in step b is a compound of Formula I. Inone embodiment the compound in step b is a compound of Formula A-I. Inone embodiment the compound in step b is a compound of Formula II. Inone embodiment the compound in step b is a compound of Formula A-II. Inone embodiment the compound in step b is a compound of Table I. In oneembodiment the compound in step b is a compound of Formula

In one embodiment the compound in step b is a compound of Formula

In one embodiment, the β-lactam antibiotic in step a is Ceftolozane orCeftazidime. In one embodiment the compound in step b is a compound ofFormula

and the β-lactam antibiotic in step a is Ceftolozane. In one embodimentthe compound in step a is b compound of Formula

and the β-lactam antibiotic in step a is Ceftolozane. In one embodiment,the invention provides a method for treating an infection in a subjectby administering a therapeutically-effective amount of one or morecompounds of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II),preferably a compound of Formula A-I or Formula A-II in conjunction witha β-lactam antibiotic, or compositions thereof. In one embodiment, themethod comprises administering to a subject in need thereof apharmaceutical composition comprising at least one of the compoundsdescribed herein, preferably a compound of Formula A-I or Formula A-IIin conjunction with a β-lactam antibiotic. In one embodiment thecompound is of Formula

in conjunction with a β-lactam antibiotic, preferably Ceftolozane orCeftazidime, or compositions thereof. In one embodiment the compound isof Formula

in conjunction with a β-lactam antibiotic, preferably Ceftolozane orCeftazidime, or compositions thereof. In one embodiment, thepharmaceutical composition can comprise any one of the compoundsdescribed herein as the sole active compound or in combination withanother compound, composition, or biological material. The compound maybe administered orally, parenterally, by inhalation, topically,rectally, nasally, buccally, vaginally, or by an implanted reservoir,external pump or catheter. The compound may be prepared for opthalmic oraerosolized uses. The compounds of the present invention can beadministered as an aerosol for the treatment of pneumonia or otherlung-based infections. In one embodiment, the aerosol delivery vehicleis an anhydrous or dry powder inhaler. One or more compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II), preferably acompound of Formula A-I or Formula A-II in conjunction with a β-lactamantibiotic, or pharmaceutical compositions thereof also may be directlyinjected or administered into an abscess, ventricle or joint. Parenteraladministration includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, cisternal, intrathecal, intrahepatic,intralesional and intracranial injection or infusion. In one embodiment,one or more compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, are administered intravenously,subcutaneously or orally. In one embodiment for administering one ormore compounds according to the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic to a cell culture, the one ormore compounds may be administered in a nutrient medium.

In one embodiment, one or more compounds according to the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or A-II in conjunction with a β-lactam antibiotic, may be used totreat a subject having a bacterial infection in which the infection iscaused or exacerbated by any type of bacteria, such as Gram-negativebacteria. In one aspect of the invention, the bacterial infection iscaused by β-lactam resistant bacteria. In one aspect the bacterialinfection is caused by β-lactamase producing bacteria. In another aspectthe bacterial infection is caused by class A, class C or class Dβ-lactamase producing bacteria. In another aspect the bacterialinfection is caused by class A β-lactamase producing bacteria. Inanother aspect the infection is caused by class C β-lactamase producingbacteria. In still another aspect the infection is caused by class Dβ-lactamase producing bacteria. In still another aspect the infection iscaused by KPC β-lactamase producing bacteria. In still another aspectthe infection is caused by OXA β-lactamase producing bacteria. In stillanother aspect, the bacterial infection is caused by a bacteria thatproduces multiple β-lactamases. Bacteria that produce multipleβ-lactamases may produce β-lactamases of the same class or of differentclasses (e.g class A and class A or class A and class C or class A andclass D etc).

Representative Gram-negative pathogens known to express β-lactamasesinclude, but are not limited to Acinetobacter spp. (includingAcinetobacter baumannii), Citrobacter spp., Escherichia spp. (includingEscherichia coli), Haemophilus influenzae, Morganella morganii,Pseudomonas aeruginosa, Klebsiella spp. (including Klebsiellapneumoniae), Enterobacter spp. (including Enterobacter cloacae andEnterobacter aerogenes), Pasteurella spp., Proteus spp. (includingProteus mirabilis), Serratia spp. (including Serratia marcescens), andProvidencia spp. Bacterial infections can be caused or exacerbated byGram-negative bacteria including strains which express β-lactamases thatmay confer resistance to penicillins, cephalosporins, monobactams and/orcarbapenems. The co-administration of a novel BLI that inhibits theseβ-lactamases with a β-lactam antibiotic could be used to treatinfections caused by β-lactam resistant bacteria.

In one aspect of the invention the infection is caused by a β-lactamaseproducing bacteria selected from Acinetobacter spp, Citrobacter spp,Escherichia coli, Enterobacter cloacae), Haemophilus influenzae,Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, andKlebsiella pneumoniae,

β-Lactam antibiotics that may be administered concurrently withcompounds of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II)include, but are not limited to cephalosporin, carbapenem, monobactam,penem and penicillin classes of antibiotics.

In one embodiment of the invention, the β-lactam antibiotic is acephalosporin. Examples of cephalosporins include, but are not limitedto, Cefacetrile (cephacetrile), Cefadroxil (cefadroxyl), Cefalexin(cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium),Cefaloridine (cephaloradine), Cefalotin (cephalothin), Cefapirin(cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin(cephazolin), Cefradine (cephradine), Cefroxadine, Ceftezole, Cefaclor,Cefamandole, Cefmetazole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil(cefproxil), Cefuroxime, Cefuzonam, Cefcapene, Cefdaloxime, Cefdinir,Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefotaxime,Cefpimizole, Cefpodoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene,Ceftizoxime, Ceftriaxone, Cefoperazone, Ceftazidime, Cefclidine,Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome,Cefaclomezine, Cefaloram, Cefaparole, Cefcanel, Cefedrolor, Cefempidone,Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole,Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, Cefuracetime,cefbuperazone, cefminox, ceforanide, cefotiam, cefpiramide, cefsulodin,ceftobiprole latamoxef, loracarbef and Ceftolozane. In one embodimentthe cephalosporin is Ceftolozane or Ceftazidime.

In one embodiment of the invention, the β-lactam antibiotic is acarbapenen. Examples of carbapenem antibiotics include, but are notlimited to, Imipenem, Imipenem/Cilastatin, Biapenem, Doripenem,Meropenem, Ertapenem and Panipenem. In one embodiment the Carbapenem isImipenem/Cilastatin or Meropenem.

In one embodiment of the invention, the β-lactam antibiotic is amonobactam. Examples of monobactam antibiotics include, but are notlimited to Aztreonam, Tigemonam, Carumonam, BAL30072 and Nocardicin A.

In one embodiment of the invention, the β-lactam antibiotic is a penem.In one embodiment of the invention, the β-lactam antibiotic is apenicillin. Examples of penicillin antibiotics include, but are notlimited to Amoxicillin, Ampicillin, Azlocillin, Mezlocillin, Apalcillin,Hetacillin, Becampicillin, Carbenicillin, Sulbenicillin, Ticarcillin,Piperacillin, Azlocillin, Mecillinam, Pivmecillinam, Methicillin,Ciclacillin, Talampicillin, Aspoxicillin, Oxacillin, Cloxacillin,Dicloxacillin, Flucloxacillin, Nafcillin and Pivampicillin.

The pharmaceutical compositions, preferably a compound of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II) in conjunction with a β-lactamantibiotic, can be used to treat a bacterial infection of any organ ortissue in the body caused by β-lactam resistant bacteria, preferably,Gram-negative β-lactam resistant bacteria. These organs or tissueinclude, without limitation, skeletal muscle, skin, bloodstream,kidneys, heart, lung and bone. For example, a pharmaceutical compositioncomprising at least one compound of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II), preferably a compound of Formula A-I or Formula A-IIin conjunction with a β-lactam antibiotic, can be administered to asubject to treat, without limitation, skin and soft tissue infections(e.g., complex skin infections), bacteremia, intra-abdominal infectionsand urinary tract infections (e.g., cUTI). In addition, a compound ofthe invention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) may be used to treatcommunity acquired respiratory infections, including, withoutlimitation, otitis media, sinusitis, chronic bronchitis and pneumonia(including community-acquired pneumonia, hospital-acquired pneumonia andventilator associated pneumonia), including pneumonia caused bydrug-resistant Pseudomonas aeruginosa. At least one compound of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II), preferably acompound of Formula A-I or Formula A-II in conjunction with a β-lactamantibiotic, can be administered to a subject to treat mixed infectionsthat comprise different types of Gram-negative bacteria, or whichcomprise both Gram-positive and Gram-negative bacteria. These types ofinfections include intra-abdominal infections andobstetrical/gynecological infections. At least one compound of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II), preferably acompound of Formula A-I or Formula A-II in conjunction with a β-lactamantibiotic, may also be administered to a subject to treat an infectionincluding, without limitation, endocarditis, nephritis, septicarthritis, intra-abdominal sepsis, bone and joint infections andosteomyelitis. At least one compound of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II), preferably compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, or pharmaceutical compositionsthereof, may also be directly injected or administered into an abscess,ventricle or joint. Pharmaceutical compositions of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II), preferably compound of Formula A-I orFormula A-II in conjunction with a β-lactam antibiotic, may beadministered as an aerosol for the treatment of pneumonia or otherlung-based infections. In one embodiment, the aerosol delivery vehicleis an anhydrous, liquid or dry powder inhaler.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of one or more compounds according to the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II), preferably a compound of Formula A-I orFormula A-II in conjunction with a β-lactam antibiotic, may be varied soas to obtain a therapeutically-effective amount of the activecompound(s) to achieve the desired therapeutic response for a particularpatient, compositions, and mode of administration. The effective amountcan be determined as described herein. The selected dosage level willdepend upon the activity of the particular compound, the route ofadministration, the severity of the condition being treated, and thecondition and prior medical history of the patient being treated.However, it is within the skill of the art to start doses of thecompound at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. In one embodiment, the data obtained fromthe assays can be used in formulating a range of dosage for use inhumans. It will be understood by one of skill in the art that the whenthe composition comprises a compound of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) and a β-lactam antibiotic, both the compound of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) and the β-lactamantibiotic are active compounds.

The method comprises administering to the subject an effective dose ofone or more compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably in conjunction with a β lactam antibiotic. Aneffective dose of a compound of the invention (e.g. compounds of FormulaI, compounds of Formula A-I, compounds of Formula II, compounds ofFormula A-II) is generally between 125 mg/day to 2000 mg/day. In oneembodiment, an effective dose is from about 0.1 to about 100 mg/kg ofone or more compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) or pharmaceutically acceptable salts thereof. In one embodiment,the dose is from about 0.1 to about 50 mg/kg of one or more compounds ofthe invention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) or pharmaceuticallyacceptable salts thereof. In another embodiment, the dose is from about1 to about 25 mg/kg of one or more compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) or pharmaceutically acceptable saltsthereof. In another embodiment, the dose is from about 1 to about 12mg/kg of one or more compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II). In another embodiment, the dose is about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 mg/kg of one or more compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II). In anotherembodiment, the compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) are administered to a human at a dose of 100 mg to 1000 mg perdose up to four times per day. In another embodiment, the compounds ofthe invention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) are administered toa human at a dose of 125 mg to 750 mg per dose up to four times per day.In another embodiment, the compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II) are administered to a human at a dose of 250 mg to 500mg per dose up to four times a day. An effective dose for cell cultureis usually between about 0.1 and about 1000 μg/mL. In one embodiment,the effect dose for cell culture is between about 0.1 and about 200μg/mL.

In one embodiment, a β-lactam antibiotic and a compound of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II) are administered in ratio of 1:4to 8:1 antibiotic:compound of the invention (e.g. compounds of FormulaI, compounds of Formula A-I, compounds of Formula II, compounds ofFormula A-II). In one embodiment the ratio is 1:4. In another embodimentthe ratio is 3:4. In another embodiment the ratio is 5:4. In anotherembodiment the ratio is 7:4. In another embodiment the ratio is 1:2. Inanother embodiment the ratio is 3:2. In another embodiment the ratio is5:2. In another embodiment the ratio is 7:2. In another embodiment theratio is 1:3. In another embodiment the ratio is 2:3. In anotherembodiment the ratio is 4:3. In another embodiment the ratio is 5:3. Inanother embodiment the ratio is 7:3. In another embodiment the ratio is1:2. In another embodiment the ratio is 3:2. In another embodiment theratio is 5:2. In another embodiment the ratio is 7:2. In anotherembodiment the ratio is 1:1. In another embodiment the ratio is 2:1. Inanother embodiment the ratio is 3:1. In another embodiment the ratio is4:1. In another embodiment the ratio is 5:1. In another embodiment theratio is 6:1. In another embodiment the ratio is 7:1. In anotherembodiment the ratio is 8:1. It will be understood by one of skill inthe art that the β-lactam antibiotic and compound of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II) can be administered within the range ofratios provided regardless of the method of drug delivery. It will alsobe understood by one of skill in the art that the β-lactam antibioticand compound of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II) can beadministered within the range of ratios provided together, for example,in a pharmaceutical composition, or sequentially, i.e. the β-lactamantibiotic is administered, followed by administration of a compound ofthe invention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II) or vice versa.

One or more compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II) may also be administered in the diet or feed of a patient oranimal. If administered as part of a total dietary intake, the amount ofcompound employed can be less than 1% by weight of the diet, such as nomore than 0.5% by weight. The diet for animals can be normal foodstuffsto which the compound can be added or it can be added to a premix.

One or more compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, can be administered as a singledaily dose or in multiple doses per day. In one embodiment, one or morecompounds of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II),preferably a compound of Formula A-I or Formula A-II in conjunction witha β-lactam antibiotic, is administered as a single dose per day. Inanother embodiment, one or more compounds of the invention (e.g.compounds of Formula I, compounds of Formula A-I, compounds of FormulaII, compounds of Formula A-II), preferably a compound of Formula A-I ofFormula A-II in conjunction with a β-lactam antibiotic is administeredas two equal doses per day. In another embodiment, the compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II), preferably acompound of Formula A-I or Formula A-II in conjunction with a β-lactamantibiotic is administered in three equal doses per day. In anotherembodiment, the compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic is administered in four equaldoses per day. The treatment regime may require administration overextended periods of time, e.g., for several days or for from two to fourweeks. The amount per administered dose or the total amount administeredwill depend on such factors as the nature and severity of the infection,the age and general health of the patient, the tolerance of the patientto the compound of the invention and the β-lactam antibiotic and themicroorganism or microorganisms involved in the infection. The treatmentregimen for one type of infection may differ greatly from the treatmentregimen of another infection. For example, one type of infection mayrequire administration via intravenous administration once daily, whileanother infection may require a treatment regimen of multiple dosingorally.

One or more compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, may be administered according tothis method until the bacterial infection is eradicated or reduced. Inone embodiment, one or more compounds of the invention (e.g. compoundsof Formula I, compounds of Formula A-I, compounds of Formula II,compounds of Formula A-II), preferably a compound of Formula A-I orFormula A-II in conjunction with a β-lactam antibiotic, are administeredfor a period of time from 3 days to 6 months. In another embodiment, oneor more compounds of the invention (e.g. compounds of Formula I,compounds of Formula A-I, compounds of Formula II, compounds of FormulaA-II), preferably a compound of Formula A-I or Formula A-II inconjunction with a β-lactam antibiotic, are administered for 7 to 56days. In another embodiment, one or more compounds of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or Formula A-II in conjunction with a β-lactam antibiotic, areadministered for 7 to 28 days. In a further embodiment, one or morecompounds of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II),preferably a compound of Formula A-I or Formula A-II in conjunction witha β-lactam antibiotic, are administered for 7 to 14 days. Compounds ofthe present invention may be administered for a longer or shorter timeperiod if it is so desired.

Other embodiments of the invention include:

A pharmaceutical composition comprising a compound of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or Formula A-II and at least 1 β-lactam antibiotic or apharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or Formula A-II and at least 1 cephalosporin antibiotic or apharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or Formula A-II and Ceftolozane or a pharmaceutically acceptablesalt thereof.

A pharmaceutical composition comprising a compound of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or Formula A-II and at least 1 carbapenem antibiotic or apharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or Formula A-II and at least 1 monobactam antibiotic or apharmaceutically acceptable salt thereof.

The embodiments described herein provide compounds of the invention(e.g. compounds of Formula I, compounds of Formula A-I, compounds ofFormula II, compounds of Formula A-II), preferably a compound of FormulaA-I or Formula A-II that are novel and active β-lactamase inhibitors.Other embodiments described herein provide novel compounds of theinvention (e.g. compounds of Formula I, compounds of Formula A-I,compounds of Formula II, compounds of Formula A-II), preferably acompound of Formula A-I or Formula A-II in conjunction with β-lactamantibiotics for treatment of infections. Further embodiments describedherein provide novel compounds of the invention (e.g. compounds ofFormula I, compounds of Formula A-I, compounds of Formula II, compoundsof Formula A-II), preferably a compound of Formula A-I or Formula A-IIthat show unexpected activity against β-lactamases that other compoundsin the class do not have.

Preparation of Compounds of the Invention

A compound of the invention (e.g. compounds of Formula I, compounds ofFormula A-I, compounds of Formula II, compounds of Formula A-II) can beprepared by a variety of synthetic routes, including synthetic schemesdescribed herein. These synthetic routes can be applied to large scalesynthesis with appropriate adjustment of reaction sequence, reactionconditions, isolation/purification methods and choice of solvents whichare environmentally friendly and cost-effective.

The following abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeaning.

-   -   Bn=benzyl    -   Boc=tert-butoxycarbonyl    -   Boc₂O=di-tert-butyldicarbonate    -   Burgess reagent=methyl N-triethylammoniumsulfonyl)carbamate    -   CDI=carbonyldiimidazole    -   CFU=colony-forming units    -   CLSI=Clinical Laboratory Standards Institute    -   cSSSI=complicated skin and skin structure infections    -   DBU=1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCM=dichloromethane    -   DEAD=diethyl azodicarboxylate    -   DIAD=diisopropyl azodicarboxylate    -   DIPEA=diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMAc=N,N-dimethylacetamide    -   DMSO=dimethyl sulfoxide    -   EDCI=1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide    -   ELSD=evaporative light scattering detector    -   EtOAc=ethyl acetate    -   ESI-MS=electrospray ionization mass spectrometry    -   Fmoc=Fluorenylmethyloxycarbonyl    -   HAP=Hospital-Acquired Pneumonia    -   HATU=2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HCl=hydrochloride    -   HOBt=1-hydroxybenzotrizole    -   Hrs=hours    -   HPLC=high performance liquid chromatography    -   Hunig's base=N,N-Diisopropylethylamine    -   Lawesson's        reagent=2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide    -   MIC=minimum inhibitory concentration    -   mL=milliliter    -   MS=mass spectrometry    -   MRSA=methicillin-resistant Staphylococcus aureus    -   NMR=nuclear magnetic resonance    -   Ns=nitrobenzenesulfonyl    -   Pa=Pseudomonas aeruginosa    -   Prep=preparative    -   Ppm=parts per million    -   Py=pyridine    -   sat.=saturated    -   rt=room temperature    -   TBAF=tetrabutylammonium fluride    -   TBS=t-butyldimethylsilyl    -   TES=triethylsilyl    -   TEA=triethylamine    -   TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy, free radical    -   THF=tetrahydrofuran    -   TFA=trifluoroacetic acid    -   TMS=trimethylsilyl    -   TLC=thin layer chromatography    -   VAP=Ventilator-Associated Pneumonia

The compounds of Formula (I) can be prepared from intermediate 1,according to the following reaction schemes and examples, ormodifications thereof, using readily available starting materials,reagents and conventional synthetic procedures including, for example,procedures described in U.S. Pat. No. 7,112,592 and WO2009/091856.Compound 3 can be synthesized following standard isoxazole ringformation chemistry from the chlorooxime intermediate 2d, which can beprepared from ester intermediate 1 (see, e.g., Abele, E.; Lukevics, E.Heterocycles 2000, 53, 2285-2336; Barr, L.; Lincoln, S. F.; Easton, C.J. Chemistry-A European Journal 2006, 12, 8571-8580; Walker, D. G.;Brodfuehrer, P. R.; Brundidge, S. P. Shih, K. M.; Sapino, C. Jr. J. Org.Chem. 1988, 53, 983-991 and references cited therein).

It may be necessary to protect certain functionalities in the moleculedepending on the nature of the R¹ group. Protecting thesefunctionalities should be within the expertise of one skilled in theart. See, e.g P. G. M. Wuts and T. W. Greene, Protective Groups inOrganic Synthesis, Fourth Edition, John Wiley and Sons, 2006, hereafterGreene.

The benzylic ether protecting group in 3 can be removed via standardhydrogenolysis conditions, such as, but not limited to, Pd/H₂ in MeOH orTHF or by acid-catalysed hydrolysis, such as, but not limited to, BCl₃in DCM to provide the hydroxy-urea intermediate 4, which can be useddirectly in the next step without further purification. Sulfation of 4can be achieved by treatment with a sulfating reagent, such a, but notlimited to, SO₃.pyridine complex, in an appropriate solvent, such aspyridine, DMF or DMAc at a temperature of 0-80° C., preferable at roomtemperature. Compound 5 can then be isolated and purified viaconventional methods. For example, 5 can be purified by standard reversephase prep-HPLC using an appropriate buffer system, i.e. ammoniumformate buffer. In some cases, 5 can be purified by normal phase silicagel chromatography after converting to an appropriate salt form, such assulfate tetrabutyl ammonium salt. The tetrabutyl ammonium salt can thenbe converted to a sodium salt by cation exchange. When a protectinggroup(s) is present in the sidechain (i.e. Boc or Fmoc for amine andguanidine protection, TBS or TES for alcohol protection, etc), adeprotection step is needed to convert 5 to its final product 6, whichcan be purified by reverse phase prep-HPLC using the conditionsmentioned above. For example, for N-Boc deprotection, 5 can be treatedwith an acid, such as TFA, in an appropriate solvent, such as DCM at atemperature of 0-30° C., preferable at 0° C. to rt to give 6. For anO-TBS, or O-TES deprotection, a fluoride reagent such as HF.pyridine,HF.NEt₃, or TBAF can be used. For Fmoc deprotection, amines, such asdiethylamine, DBU, piperidine, etc can be used.

EXAMPLES

The specific examples which follow illustrate the synthesis of certaincompounds. The methods disclosed may be adopted to variations in orderto produce compounds of Formula (I), but not otherwise specificallydisclosed. Further, the disclosure includes variations of the methodsdescribed herein to produce the compounds of Formula (I) that would beunderstood by one skilled in the art based on the instant disclosure.

All temperatures are understood to be in Centigrade (C) when notspecified. The nuclear magnetic resonance (NMR) spectral characteristicsrefer to chemical shifts (γ) expressed in parts per million (ppm) versustetramethylsilane (TMS) as reference standard. The relative areareported for the various shifts in the proton NMR spectral datacorresponds to the number of hydrogen atoms of a particular functionaltype in the molecule. The nature of the shifts as to multiplicity isreported as broad singlet (br s), broad doublet (br d), singlet (s),multiplet (m), doublet (d), quartet (q), doublet of doublet (dd),doublet of triplet (dt), and doublet of quartet (dq). The solventsemployed for taking NMR spectra are DMSO-d6(perdeuterodimethysulfoxide), D₂O (deuterated water), CDCl₃(deuterochloroform) and other conventional deuterated solvents. Theprep-HPLC conditions are: Waters SunFire® C18 (30×100 mm, 5 μm OBD)column; flow rate: 30-80 mL/minute, ELSD or Mass-triggered fractioncollection; sample loading: Each injection loading varied from 30-300 mgfor different crude samples depending on their solubility and purityprofiles; Solvent system using ammonium formate buffer: solvent A: waterwith 20 mM ammonium formate, solvent B: 85% of acetonitrile in waterwith 20 mM ammonium formate. Solvent system using NH₄HCO₃ buffer:solvent A: water with 10 mM NH₄HCO₃, solvent B: acetonitrile. Solventsystem using NH₄OH buffer: solvent A: water with 0.1% NH₄OH, solvent B:acetonitrile with 0.1% NH₄OH.

Example 1 Synthesis of (2S,5R)-ethyl6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(Intermediate Compound 1) c

Step 1: Synthesis of (S)-1-tert-butyl 2-ethyl5-oxopiperidine-1,2-dicarboxylate Method A

n-BuLi (600 mL, 1.5 mol) was added dropwise to a solution of TMSCHN₂(690 mL, 1.38 mol) in dry THF (3 L) at −78° C., and the mixture wasstirred at −78° C. for 30 minutes. The mixture was then transferred to asolution of (S)-1-tert-butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate(300 g, 1.17 mol) in dry THF (3 L) via cannula, and the mixture wasstirred at −78° C. for 30 minutes. The reaction mixture was thenquenched with sat. NH₄Cl solution, and extracted with DCM (3×). Thecombined organic layer was concentrated under reduced pressure and thecrude product was purified by silica gel column chromatography (3:1petroleum ether/EtOAc) to afford (S)-ethyl2-((tert-butoxycarbonyl)amino)-6-diazo-5-oxohexanoate (262 g, 75%) as ayellow solid.

A solution of (S)-ethyl2-((tert-butoxycarbonyl)amino)-6-diazo-5-oxohexanoate (350 g, 1.18 mol)in DCM (1500 mL) was added to a 0° C. solution of Rh₂(OAc)₄ (3.5 g, 7.9mmol) in DCM (750 mL). The reaction mixture was then stirred at 20° C.overnight and then concentrated under reduced pressure. The crude samplewas purified by silica gel column chromatography (5:1 petroleumether/EtOAc) to afford (5)-1-ten-butyl 2-ethyl5-oxopiperidine-1,2-dicarboxylate (175.9 g, 55%) as a yellow oil.

Method B

t-BuOK (330 g, 2.9 mol) was added to a solution of trimethylsulfoxoniumiodide (750 g, 3.5 mol) in dry DMSO (3 L) and the mixture was stirred atrt for 1 h. (S)-1-tert-Butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate(900 g, 3.5 mol) was added and the mixture was stirred at rt for 2-3hrs. Water was added to quench the reaction and the mixture wasextracted with EtOAc (5×). The combined organic layer was concentratedunder reduced pressure and the crude sample was purified by silica gelcolumn chromatography (1:1 petroleum ether/EtOAc then 1:10 MeOH/DCM) toafford sulfoxonium ylide intermediate (977 g, 80%) as a white solid.

A solution of sulfoxonium ylide intermediate (156 g, 0.446 mol) and[Ir(COD)Cl]₂ (3 g, 4.46 mmol) in toluene (4 L) was degassed by bubblingnitrogen through the solution for 10 minutes. The reaction mixture washeated to 80-90° C. for 2-3 hrs and then cooled to 20° C. The toluenewas concentrated under reduced pressure and the resulting residue waspurified by silica gel column chromatography (10:1 to 3:1 gradientelution petroleum ether/EtOAc) to afford (S)-1-tert-butyl 2-ethyl5-oxopiperidine-1,2-dicarboxylate (140 g, 57.8%) as a yellow oil.

Step 2: Synthesis of (2S,5S)-1-tert-butyl 2-ethyl5-hydroxypiperidine-1,2-dicarboxylate

NaBH₄ (36 g, 1.0 mol) was added in portions to a −40° C. solution of(S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (250 g, 0.92mol) in EtOH (1500 mL). The reaction mixture was then stirred at −40° C.for 0.5 hr then quenched with 10% HOAc solution. After diluting withwater, the mixture was extracted with DCM (3×). The combined organiclayer was concentrated under reduced pressure and purified by silica gelcolumn chromatography (1:1 petroleum ether/EtOAc) to afford(2S,5S)-1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (205g, 80%) as a yellow oil.

Step 3: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate

A solution of 2-nitrobenzene-1-sulfonyl chloride (500 g, 2.26 mol) inpyridine (1500 mL) was added dropwise to a 0° C. solution ofO-benzylhydroxylamine hydrochloride (400 g, 2.51 mol) in pyridine (1500mL). The reaction mixture was allowed to warm to room temperature thenwas stirred at 20° C. overnight. The mixture was concentrated underreduced pressure, diluted with DCM and washed with HCl (10%, 3×). Thecombined organic layer was concentrated under reduced pressure andre-crystallized with DCM to affordN-(benzyloxy)-2-nitrobenzenesulfonamide (485 g, 62.6%) as a yellowsolid.

To a solution of N-(benzyloxy)-2-nitrobenzenesulfonamide (212 g, 0.69mol) in THF (1000 mL) was added (2S,5S)-1-tert-butyl 2-ethyl5-hydroxypiperidine-1,2-dicarboxylate (171 g, 0.63 mol) and PPh₃ (275 g,1.05 mol), followed by dropwise addition of a solution of DEAD (195 g,1.12 mol) in THF (500 mL). The mixture was then stirred at 20° C.overnight. The reaction mixture was then concentrated under reducedpressure and purified by silica gel column chromatography (3:1 petroleumether/EtOAc) to afford (2S,5R)-1-tert-butyl 2-ethyl5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate(283.8 g, 80%) as a yellow oil.

Step 4: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl5-((benzyloxy)amino)piperidine-1,2-dicarboxylate

LiOH.H₂O (95 g, 2.3 mol) and 2-mercaptoacetic acid (124 g, 1.3 mol) wereadded to a solution of (2S,5R)-1-tert-butyl 2-ethyl5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1,2-dicarboxylate(251 g, 0.45 mol) in DMF (1200 mL). The reaction mixture was thenstirred at 20° C. overnight. The reaction mixture was diluted with waterand extracted with EtOAc (3×). The combined organic layer was washedwith saturated sodium chloride (3×), concentrated under reduced pressureand purified by silica gel column chromatography (3:1 petroleumether/EtOAc) to afford (2S,5R)-1-tert-butyl 2-ethyl5-((benzyloxy)amino)piperidine-1,2-dicarboxylate (122.9 g, 85%) as ayellow solid.

Step 5: Synthesis of (2S,5R)-ethyl5-((benzyloxy)amino)piperidine-2-carboxylate

TFA (600 mL) was added to a solution of (2S,5R)-1-tert-butyl 2-ethyl5-((benzyloxy)amino)piperidine-1,2-dicarboxylate (263 g, 0.7 mol) in DCM(600 mL) at 20° C. The mixture was stirred at rt overnight and thenconcentrated under reduced pressure. The crude product was adjusted topH 10 with sat. NaHCO₃ solution then extracted with DCM (3×). Thecombined organic layer was concentrated under reduced pressure andpurified by silica gel column chromatography (20:1 DCM/MeOH) to afford(2S,5R)-ethyl 5-((benzyloxy)amino)piperidine-2-carboxylate (184.9 g,95%) as a yellow oil.

Step 6: Synthesis of (2S,5R)-ethyl6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate

Triphosgene (21.3 g, 72 mmol) was added in portions to a 0° C. solutionof (2S,5R)-ethyl 5-((benzyloxy)amino)piperidine-2-carboxylate (50 g,0.18 mol) and DIPEA (128 mL, 0.72 mol) in DCM (2000 mL). The reactionmixture was allowed to warm to rt. After stirring at rt overnight, thereaction mixture was washed with H₃PO₄ (10%), sat. NaHCO₃ and saturatedNaCl. The combined organic layer was concentrated under reduced pressureand purified by silica gel column chromatography (3:1 petroleumether/EtOAc) to afford (2S,5R)-ethyl6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (27.4 g,50%) as a yellow solid. ¹H NMR (400 Mz, CDCl₃): δ 7.43-7.36 (m, 5H),5.06 (d, J=11.4 Hz, 1H), 4.90 (d, J=11.4 Hz, 1H), 4.24 (q, J=7.1 Hz,2H), 4.11-4.08 (m, 1H), 3.32-3.31 (m, 1H), 3.08-3.05 (m, 1H), 2.93 (d,J=11.9 Hz, 1H), 2.14-2.05 (m, 2H), 2.05-2.00 (m, 1H), 1.71-1.63 (m, 1H),1.29 (t, J=7.1 Hz, 3H).

Example 2 Synthesis of(2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde(Intermediate Compound 2b)

LiBH₄ (0.54 g, 24.67 mmol) was added to a −10° C. solution of(2S,5R)-ethyl6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (5 g,16.44 mmol) in MeOH (50 mL). After 15 minutes another portion of LiBH₄(0.54 g, 24.67 mmol) was added and the mixture was stirred at −10 to 0°C. for 4-5 h. The reaction mixture was carefully quenched by addition ofsat. NaH₂PO₄ (50 mL) at 0° C. The mixture was diluted with water (20 mL)and extracted with DCM (3×). The combined organic layer was concentratedand purified by silica gel column chromatography (gradient elution0-100% EtOAc/petroleum ether, then 0-2% MeOH/EtOAc) to give(2S,5R)-6-(benzyloxy)-2-(hydroxymethyl)-1,6-diazabicyclo[3.2.1]octan-7-one(3.8 g, 88%) as a white solid. ESI-MS (EI⁺, m/z): 263.1. ¹H NMR (500M,CDCl₃): 7.44-7.35 (m, 5H), 5.05 (d, J=11.5 Hz, 1H), 4.90 (d, J=11.5 Hz,1H), 3.73-3.69 (m, 1H), 3.61-3.58 (m, 2H), 3.33 (m, 1H), 3.01 (br d,J=12.0 Hz, 1H), 2.91 (m, 1H), 2.03-1.95 (m, 2H), 1.58-1.54 (m, 1H),1.39-1.24 (m, 1H).

TEMPO (48 mg, 0.3 mmol) was added in portions to a 0° C. solution of(2S,5R)-6-(benzyloxy)-2-(hydroxymethyl)-1,6-diazabicyclo[3.2.1]octan-7-one(7.8 g, 30 mmol) and 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione (7.0g, 30 mmol) in DCM (100 mL). The mixture was stirred at 0° C. for 2 h,and filtered through Celite®. The filtrate was dried over Na₂SO₄ andconcentrated to afford(2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde(7.0 g, 90%) as a yellow oil. ESI-MS (Er, m/z): 261.1. ¹H NMR (500M,CDCl₃): 9.74 (s, 1H), 7.45-7.36 (m, 5H), 5.07 (d, J=11.5 Hz, 1H), 4.92(d, J=11.5 Hz, 1H), 3.89 (d, J=8.0 Hz, 1H), 3.27 (m, 1H), 3.21-3.05 (m,1H), 2.56 (d, J=12.0 Hz, 1H), 2.20-2.15 (m, 1H), 2.05-2.01 (m, 1H),1.95-1.93 (m, 1H), 1.49-1.46 (m, 1H).

Example 3 Synthesis of(E)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehydeoxime (Intermediate Compound 2c)

A solution of(2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehyde(510 mg, 1.96 mmol), hydroxylamine hydrochloride (158 mg, 2.27 mmol) andpyridine (621 mg, 7.85 mmol) in EtOH (15 mL) was stirred at rt for 2hrs. Then, the reaction mixture was concentrated and the residue wasdiluted with DCM (25 mL), washed with water (3×), and saturated sodiumchloride, dried over Na₂SO₄ and concentrated. The residue was purifiedby silica gel column chromatography (3:1 to 3:2 petroleum ether/EtOAC)to afford(E)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbaldehydeoxime (228 mg, 42%) as a white solid. ESI-MS (EI⁺, m/z): 276 [M+H]⁺.

Example 4 Synthesis of(2S,5R)-2-(5-(aminomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 601)

Step 1

NCS (295 mg, 2.2 mmol) was added to a solution of6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbaldehyde oxime(560 mg, 2.0 mmol) in dry DCM (15 mL) at rt. Pyridine (one drop) wasthen added, and the reaction mixture was stirred at rt for 18 hrs. Thesolution was evaporated to afford(2S,5R)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbimidoylchloride, which was directly used in the next step. ESI-MS (EI⁺, m/z):274 [M-Cl]⁺.

Step 2

tert-Butyl prop-2-yn-1-ylcarbamate (0.37 g, 2.4 mmol) was added to thesolution of(2S,5R)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbimidoylchloride (˜2.0 mmol) in dry DCM (20 mL), followed by the addition of TEA(0.31 mL, 2.2 mmol) in dry DCM (2.0 mL) over a period of 30 minutes. Thereaction mixture was stirred at rt overnight, then the mixture wasdiluted with EtOAc, washed with water and saturated sodium chloride. Theorganic layer was dried over Na₂SO₄, concentrated and purified by silicagel column chromatography (1:2 petroleum ether/EtOAc) to affordtert-butyl((3-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methyl)carbamate(180 mg, 21% for two steps) as a white solid. ESI-MS (EI⁺, m/z): 429.1[M+H]⁺. ¹H NMR (500 MHz, CDCl₃): δ 7.45-7.35 (m, 5H), 6.24 (s, 1H), 5.09(d, J=11.5 Hz, 1H), 4.93 (d, J=11.5 Hz, 1H), 4.58 (d, J=7.5 Hz, 1H),4.43 (m, 2H), 3.32 (s, 1H), 2.86-2.84 (m, 1H), 2.68 (d, J=11.5 Hz, 1H),2.34-2.30 (m, 1H), 2.23-2.18 (m, 1H), 2.10-2.04 (m, 1H), 1.82-1.78 (m,1H), 1.45 (s, 9H).

Step 3

To a solution of tert-butylβ-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methylcarbamate(210 mg, 0.5 mmol) in THF (5 mL) was added 10% Pd/C (100 mg). Thereaction mixture was then filtered and concentrated to affordtert-butyl((3-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methyl)carbamate(180 mg, 99%) as a light yellow solid, which was used directly in thenext step. ESI-MS (EI⁺, m/z): 339.1 [M+H]⁺.

Step 4

To a solution of tert-butylβ-((2S,5R)-6-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)isoxazol-5-yl)methylcarbamate(180 mg, 0.5 mmol) in dry pyridine (2.5 mL) was added SO₃.Py (480 mg,3.0 mmol). The mixture was stirred at rt for 3 hrs and then concentratedunder reduced pressure. The residue was re-dissolved in aqueous NaH₂PO₄(1.5 M, 20 mL) then tetrabutylammonium hydrogensulphate (230 mg, 0.67mmol) was added. The mixture was stirred at rt for 20 minutes thenextracted with EtOAc (4×). The combined organic layer was dried andconcentrated and the residue was purified by silica gel columnchromatography (gradient elution 10:1 to 3:1 DCM/acetone) to affordtetrabutylammonium(2S,5R)-2-(5-(((tert-butoxycarbonyl)amino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylsulfate (180 mg, 54%) as a white solid. ESI-MS (EI⁻, m/z): 417.0 [M−H]⁻.¹H NMR (500 MHz, CDCl₃): δ 6.28 (s, 1H), 4.97 (bs, 1H), 4.54 (d, J=7 Hz,1H), 4.44-4.30 (m, 2H), 4.35 (m, 1H), 3.35-3.28 (m, 8H), 3.19-3.17 (m,1H), 2.76 (d, J=11.5 Hz, 1H), 2.36-2.32 (m, 1H), 2.27-2.24 (m, 1H),2.18-2.12 (m, 1H), 1.87-1.81 (m, 1H), 1.71-1.65 (m, 8H), 1.50-1.47 (m,17H), 1.01 (t, J=7.0 Hz, 12H).

Step 5, Resin Exchange

Tetrabutylammonium(2S,5R)-2-(5-(tert-butoxycarbonylaminomethyl)-isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-ylsulfate (180 mg, 0.27 mmol) was dissolved in a minimum amount of HPLCgrade water (˜10 mL) and passed through a column of 20 g of DOWEX 50WX 8Na⁺ resin (the resin was pre-washed with >0.5 L of HPLC grade water) andeluted with HPLC grade water to afford sodium(2S,5R)-2-(5-(((tert-butoxycarbonyl)amino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylsulfate (109 mg, 92%) was obtained after lyophilization as a whitesolid. ESI-MS (EI⁻, m/z): 417.0 [M−H]⁻. ¹H NMR (500 MHz, D₂O): δ6.32 (s,1H), 4.61 (d, J=6.5 Hz, 1H), 4.32 (s, 2H), 4.13 (m, 1H), 3.10-3.08 (m,1H), 2.91 (d, J=12.5 Hz, 1H), 2.21-2.17 (m, 1H), 2.09-2.02 (m, 2H),1.86-1.82 (m, 1H), 1.34 (s, 9H).

Step 6

TFA (0.40 mL) was added to a 0° C. solution of sodium(2S,5R)-2-(5-(tert-butoxycarbonylaminomethyl)-isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-ylsulfate (54 mg, 0.12 mmol) in dry DCM (1.2 mL). The reaction mixture wasstirred at 0° C. for 30 minutes to 1 h and then diluted with diethylether. The precipitate was collected via centrifugation, washed withether (3×) and further dried under high vacuum to provide(2S,5R)-2-(5-(aminomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate as a TFA salt (˜30 mg). ESI-MS (EI⁺, m/z): 319.2. TheTFA salt (˜30 mg) was further purified by prep-HPLC using ammoniumformate buffer to provide(2S,5R)-2-(5-(aminomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (601, 10 mg, 26%) as a white solid. ESI-MS (EI⁺, m/z):319.21. ¹H NMR (300 MHz, D₂O) δ 6.61 (s, 1H), 4.68-4.65 (m, 1H), 4.35(s, 2H), 4.17-4.14 (m, 1H), 3.14-3.10 (m, 1H), 2.95-2.91 (m, 1H),2.31-1.80 (m, 4H).

Example 5 Synthesis of(2S,5R)-2-(5-(guanidinomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 603)

Step 1: Synthesis of1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne:

Di-tert-butyl(1H-pyrazol-1-yl)methanediylidenedicarbamate (1.71 g, 5.5mmol) was added to a 0° C. solution of prop-2-yn-1-amine (275 mg, 5.0mmol) and TEA (1.5 g, 15 mmol) in MeOH (40 mL). The reaction mixture wasstirred at 0° C. for 3 hrs and then concentrated under reduced pressure.The residue was purified by silica gel column chromatography (1:10EtOAc/Hexanes) to give 1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne (1.4 g, 93%) as a white solid. ESI-MS (EI⁺, m/z): 298.1[M+H]⁺.

Step 2: Synthesis of(2S,5R)-6-(benzyloxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one

1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne (362 mg, 1.22 mmol)was added to the solution of(2S,5R)-6-(benzyloxy)-N-hydroxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbimidoylchloride (473 mg, 1.53 mmol) in dry DCM (20 mL) at rt, followed bydropwise addition of TEA (0.16 g, 1.53 mmol). The mixture was stirred atrt for 2 hrs and then concentrated. The residue was purified by silicagel column chromatography (gradient elution 1:15 to 1:5 EtOAc/Hexanes)to give(2S,5R)-6-(benzyloxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one(190 mg, 22% for two steps) as a white solid. ESI-MS (EI⁺, m/z): 571.2[M+H]⁺. ¹H NMR (500 MHz, CDCl₃): δ 11.49 (s, 1H), 8.86 (bs, 1H),7.47-7.34 (m, 5H), 6.32 (s, 1H), 5.09 (d, J=11.5 Hz, 1H), 4.93 (d,J=12.0 Hz, 1H), 4.87-4.76 (m, 2H), 4.59 (d, J=7.0 Hz, 1H), 3.36 (s, 1H),2.87-2.85 (m, 1H), 2.70 (d, J=12 Hz, 1H), 2.35-2.30 (m, 1H), 2.24-2.18(m, 1H), 2.10-2.05 (m, 1H), 1.81-1.78 (m, 1H), 1.50 (s, 18H).

Step 3: Synthesis of(2S,5R)-6-(hydroxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one

To a solution of(2S,5R)-6-(benzyloxy)-2-(5-(2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one(240 mg, 0.42 mmol) in THF (20 mL) was added 10% Pd/C (120 mg). Themixture was stirred under H₂ atmosphere at rt for 1.5 hrs. The reactionmixture was then filtered and concentrated to afford(2S,5R)-6-(hydroxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one(200 mg, 99%) as white solid, which was directly used in the next step.ESI-MS (EI⁺, m/z): 481.2 [M+H]⁺.

Step 4: Synthesis oftetrabutylammonium(2S,5R)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-ylsulfate

To a solution of(2S,5R)-6-(hydroxy)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one(200 mg, 0.42 mmol) in dry pyridine (2.5 mL) was added SO₃.Py (400 mg,2.5 mmol). The mixture was stirred at rt for 3 hrs then concentratedunder reduced pressure. The residue was re-dissolved in aqueous NaH₂PO₄(1.5 M, 20 mL). Tetrabutylammonium hydrogensulphate (200 mg, 0.58 mmol)was added. The mixture was stirred at rt for 20 minutes then wasextracted with EtOAc (4×). The combined organic layer was dried andconcentrated and the residue was purified by silica gel columnchromatography (gradient elution 10:1 to 5:1 DCM/acetone) to givetetrabutylammonium(2S,5R)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-ylsulfate (220 mg, 66%) as a white solid. ESI-MS (EI⁻, m/z): 559.0 [M−H]⁻.¹H NMR (500 MHz, CDCl₃): δ 11.48 (s, 1H), 8.78 (bs, 1H), 6.34 (s, 1H),4.77-4.76 (m, 2H), 4.55 (d, J=7.5 Hz, 1H), 4.35 (m, 1H), 3.38-3.28 (m,8H), 3.20-3.18 (m, 1H), 2.78 (d, J=11.5 Hz, 1H), 2.37-2.30 (m, 1H),2.28-2.24 (m, 1H), 2.17-2.11 (m, 1H), 1.88-1.82 (m, 1H), 1.71-1.63 (m,8H), 1.50-1.44 (m, 26H), 1.01 (t, J=7.0 Hz, 12H).

Step 5

TFA (2.20 mL) was added to a 0° C. solution of tetrabutylammonium(2S,5R)-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-ylsulfate (465 mg, 0.60 mmol) in dry DCM (6.60 mL). The reaction mixturewas allowed to warm to rt. The reaction mixture was stirred at rt for 2hrs and then diluted with ether. The precipitate was collected viacentrifugation, washed with diethyl ether (3×) and further dried underhigh vacuum. The crude TFA salt was purified by prep-HPLC using ammoniumformate buffer to provide(2S,5R)-2-(5-(guanidinomethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (603, 60 mg, 20%) as a white solid. ESI-MS (EI⁺, m/z):361.2. ¹H NMR (300 MHz, D₂O/DMSO-d6) δ 6.58 (s, 1H), 4.74-4.71 (m, 1H),4.67 (s, 2H), 4.23 (br s, 1H), 3.22-3.17 (m, 1H), 2.99 (d, J=12.0 Hz,1H), 2.36-2.26 (m, 1H), 2.24-2.10 (m, 2H), 2.02-1.91 (m, 1H).

Example 6 Synthesis of(2S,5R)-2-(5-(2-aminoethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 602)

Following Steps 1-6 in Example 4, replacing tert-butylprop-2-yn-1-ylcarbamate in Step 2 with tert-butylbut-3-yn-1-ylcarbamate;(2S,5R)-2-(5-(2-aminoethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate TFA salt (602, 8 mg) was obtained as a light yellowsolid. ESI-MS (EI⁺, m/z): 333.2. ¹H NMR (300 MHz, D₂O) δ 6.39 (s, 1H),4.62 (d, J=6.8 Hz, 1H), 4.16-4.12 (m, 1H), 3.32 (t, J=7.1 Hz, 3H),3.25-3.04 (m, 4H), 2.97-2.93 (m, 1H), 2.30-1.79 (m, 4H).

Example 7 Synthesis of(2S,5R)-2-(5-(2-guanidinoethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 604)

Following Steps 1-5 in Example 5, replacing prop-2-yn-1-amine in Step 1with but-3-yn-1-amine;(2S,5R)-2-(5-(2-guanidinoethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (604, 48 mg) was obtained as a light yellow solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):375.1. ¹H NMR (300 MHz, D₂O/DMSO-d6) δ 6.33 (s, 1H), 4.62 (d, J=6.6 Hz,1H), 4.14 (br s, 1H), 3.51 (t, J=6.4 Hz, 2H), 3.15-3.07 (m, 1H), 3.05(t, J=6.4 Hz, 2H), 2.88 (d, J=6.4 Hz, 1H), 2.26-1.99 (m, 3H), 1.85-1.80(m, 1H).

Example 8 Synthesis of(2S,5R)-7-oxo-2-(5-(piperidin-4-yl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 606)

Following Steps 1-6 in Example 4, replacing tert-butylprop-2-yn-1-ylcarbamate in Step 2 with tert-butyl4-ethynylpiperidine-1-carboxylate,(2S,5R)-7-oxo-2-(5-(piperidin-4-yl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (606, 35 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):371.3.

Example 9 Synthesis of(2S,5R)-2-(5-(1-carbamimidoylpiperidin-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 607)

Following Steps 1-5 in Example 5, replacing prop-2-yn-1-amine in Step 1with 4-ethynylpiperidine;(2S,5R)-2-(5-(1-carbamimidoylpiperidin-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate TFA salt (607, 32 mg) was obtained. ESI-MS (EI⁺, m/z):415.2.

Example 10 Synthesis of(2S,5R)-2-(5-((2-aminoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 605)

Following Steps 1-6 in Example 4, replacing tert-butylprop-2-yn-1-ylcarbamate in Step 2 withtert-butyl(2-propiolamidoethyl)carbamate,(2S,5R)-2-(5-((2-aminoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate TFA salt (605, 20 mg) was obtained as a light yellowsolid. ESI-MS (EI⁺, m/z): 376.1. ¹H NMR (300 MHz, D₂O) δ 7.05 (s, 1H),4.70 (m, 1H, overlapped with D₂O peak) 4.16 (br s, 1H), 3.66 (t, J=5.8Hz, 2H), 3.19 (t, J=5.8 Hz, 2H), 3.16-3.08 (m, 1H), 2.93 (d, J=12.0 Hz,1H), 2.31-2.24 (m, 1H), 2.16-2.04 (m, 2H), 1.94-1.85 (m, 1H).

Example 11 Synthesis of(2S,5R)-2-(5-((2-guanidinoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 608)

Following Steps 1-5 in Example 5, replacing prop-2-yn-1-amine in Step 1with N-(2-aminoethyl)propiolamide;(2S,5R)-2-(5-((2-guanidinoethyl)carbamoyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (608, 25 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):418.12. ¹H NMR (300 MHz, D₂O) δ 4.79-4.76 (m, 1H), 4.16 (br s, 1H),4.47-4.32 (m, 4H), 4.17-4.11 (m, 2H), 3.19-3.16 (m, 1H), 2.92 (d, J=12.0Hz, 1H), 2.32-2.22 (m, 1H), 2.20-2.06 (m, 2H), 1.97-1.88 (m, 1H).

Example 12 Synthesis of(2S,5R)-7-oxo-2-(5-(2-(piperidin-4-ylamino)ethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 609)

Synthetic Scheme:

Procedures and Characterization Step 1: Synthesis of tert-butyl4-(but-3-ynylamino)piperidine-1-carboxylate

A mixture of but-3-yn-1-amine hydrochloride (26.4 g, 0.25 mol), K₂CO₃(17.4 g, 0.13 mol), and tert-butyl 4-oxopiperidine-1-carboxylate (41.8g, 0.21 mol) in MeOH (500 mL) was stirred at rt for 5 hrs. Then,NaBH(OAc)₃ (133.6 g, 0.63 mol) was added and the suspension was stirredat rt for 17 hrs. The crude reaction mixture was used directly in thenext step. ESI-MS (EI⁺, m/z): 253.2 [M+H]⁺.

Step 2: Synthesis of tert-butyl4-(but-3-ynyl(tert-butoxycarbonyl)amino)piperidine-1-carboxylate

The reaction mixture in Step 1 was cooled to 0° C. and Et₃N (88.0 mL,0.63 mol) and (Boc)₂O (91.6 g, 0.42 mol) were added. The solution wasallowed to warm to rt, then was stirred at rt for 17 hrs. The reactionmixture was concentrated, and then EtOAc (800 mL) was added. The organiclayer was washed with saturated sodium chloride (3×), dried over Na₂SO₄,and concentrated. The residue was purified by silica gel columnchromatography (gradient elution 0˜10% EtOAC/petroleum ether) to affordtert-butyl4-(but-3-ynyl(tert-butoxycarbonyl)amino)piperidine-1-carboxylate (66.0g, 90%) as a colorless oil. ESI-MS (EI⁺, m/z): 353.2 [M+H]⁺.

Step 3-6

Following Steps 2-5 in Example 5, replacing1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne in Step 2 withtert-butyl4-(but-3-yn-1-yl(tert-butoxycarbonyl)amino)piperidine-1-carboxylate;(2S,5R)-7-oxo-2-(5-(2-(piperidin-4-ylamino)ethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (609, 220 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):416.4. ¹H NMR (300 MHz, D₂O) δ 6.29 (s, 1H), 4.59 (d, J=6.3 Hz, 1H),4.12 (s, 1H), 3.39 (d, J=13.3 Hz, 2H), 3.10-2.91 (m, 9H), 2.23-1.95 (m,5H), 1.87-1.76 (m, 1H), 1.54-1.40 (m, 2H).

Example 13 Synthesis of(2S,5R)-2-(5-(2-((1-carbamimidoylpiperidin-4-yl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 610)

Synthetic Scheme:

Procedures and Characterization Step 1: Synthesis of piperidin-4-one

A mixture of tert-butyl 4-oxopiperidine-1-carboxylate (10.0 g, 50 mmol),TFA (20 mL) and CH₂Cl₂ (100 mL) was stirred at rt for 1 h. The reactionmixture was then concentrated to afford piperidin-4-one (15.0 g, 100%)as a colorless oil. ¹H NMR: (500 MHz, DMSO-d₆): δ 2.51 (m, 4H), 3.43 (m,4H), 9.03 (m, 2H).

Step 2: Synthesis ofdi-tert-butyl(4-oxopiperidin-1-yl)methylenedicarbamate

(Z)-di-tert-butyl(1H-imidazol-1-yl)methylenedicarbamate (16.3 g, 52.5mmol) was add to a 0° C. solution of piperidin-4-one (15 g, 50 mmol) andEt₃N (25.4 g, 251 mmol) in MeOH (200 mL). The reaction mixture wasallowed to warm to rt then was stirred at rt for 17 hrs. The reactionmixture was concentrated, and then EtOAc (300 mL) was added. The organiclayer was washed with saturated sodium chloride (3×), dried over Na₂SO₄,and concentrated. The residue was purified by silica gel columnchromatography (gradient elution 0˜20% EtOAc/petroleum ether) to afforddi-tert-butyl(4-oxopiperidin-1-yl)methylenedicarbamate (12.8 g, 75%) asa white solid. ESI-MS (EI⁺, m/z): 342.1 [M+H]⁺.

Step 3: Synthesis ofdi-tert-butyl(4-(but-3-ynylamino)piperidin-1-yl)methanediylidenedicarbamate

A mixture of but-3-yn-1-amine hydrochloride (3.0 g, 28.4 mmol), K₂CO₃(2.0 g, 14.2 mmol), MeOH (150 mL),di-tert-butyl(4-oxopiperidin-1-yl)methylene-dicarbamate (8.1 g, 23.7mmol) and 4 Å molecular sieve (6.0 g) was stirred at rt for 3 hrs. Then,NaBH(OAc)₃ (15.1 g, 71.1 mmol) was added, and the suspension was stirredat 70° C. for 1 h. The reaction mixture was filtered and concentrated toafforddi-tert-butyl(4-(but-3-ynylamino)piperidin-1-yl)methanediylidenedicarbamateas a yellow oil (˜15.0 g), which was used directly in the next step.

Step 4: Synthesis ofdi-tert-butyl(4-(but-3-ynyl(tert-butylcarbamate))piperidin-1-yl)methanedi-ylidenedicarbamate

A mixture of di-tert-butyl(4-(but-3-ynylamino)piperidin-1-yl)methanediylidenedicarbamate (˜15.0 g), di-tert-butyl dicarbonate (10.3g, 47.4 mmol), aq. NaHCO₃ (60 mL) in THF (120 mL) was stirred at rt for2 hrs. The reaction mixture was then concentrated and extracted withEtOAc (3×). The combined organics were dried (Na₂SO₄) and concentrated.The residue was purified by silica gel column chromatography (gradientelution 0-20% EtOAc/petroleum ether) to afforddi-tert-butyl(4-(but-3-ynyl(tert-butylcarbamate))piperidin-1-yl)methanedi-ylidenedicarbamateas a colorless oil (7.1 g, 60%). ESI-MS (EI⁺, m/z): 495.3 [M+H]⁺.

Step 5-8

Following Steps 2-5 in Example 5, replacing1-(2,3-bis(tert-butoxycarbonyl)guanidino)prop-2-yne in Step 2 withtert-butyl(1-(N,N′-bis(tert-butoxycarbonyl)carbamimidoyl)piperidin-4-yl)(but-3-yn-1-yl)carbamate;(2S,5R)-2-(5-(2-((1-carbamimidoylpiperidin-4-yl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (610, 502 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):458.0. ¹H NMR (300 MHz, D₂O) δ 6.35 (s, 1H), 4.59 (d, J=7.0 Hz, 1H),4.12 (s, 1H), 3.85 (d, J=14.5 Hz, 2H), 3.40-3.30 (m, 3H), 3.20-2.90 (m,6H), 2.20-1.75 (m, 6H), 1.62-1.46 (m, 2H).

The Compounds Described in Examples 14-33 were Prepared as Desribed inthe Reaction Schemes Following Similar Procedures of Examples 1-13Example 14 Synthesis of(2S,5R)-2-(5-((R*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 611)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((R*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (611, 46 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):391.0. ¹H NMR (300 MHz, D₂O) δ 6.46 (s, 1H), 5.04 (t, J=5.2 Hz, 1H),4.62 (d, J=6.7 Hz, 1H), 4.11 (s, 1H), 3.56 (d, J=5.2 Hz, 2H), 3.08 (d,J=11.6 Hz, 1H), 2.85 (d, J=12.1 Hz, 1H), 2.24-2.14 (m, 1H), 2.12-2.00(m, 2H), 1.86-1.76 (m, 1H).

Example 15 Synthesis of(2S,5R)-2-(5-((S*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 612)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((S*)-2-guanidino-1-hydroxyethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (612, 72 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):391.0. ¹H NMR (300 MHz, D₂O) δ 6.47 (s, 1H), 5.04 (t, J=5.4 Hz, 1H),4.63 (d, J=6.2 Hz, 1H), 4.12 (s, 1H), 3.65-3.49 (m, 2H), 3.09 (br d,J=12.7 Hz, 1H), 2.87 (d, J=12.2 Hz, 1H), 2.20-1.98 (m, 3H), 1.86-1.78(m, 1H).

Example 16 Synthesis of(2S,5R)-2-(5-((1-guanidinocyclopropyl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 613)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1-guanidinocyclopropyl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (613, 28 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):401.3. ¹H NMR (300 MHz, D₂O) δ 6.35 (s, 1H), 4.61 (d, J=6.6 Hz, 1H),4.12 (br s, 1H), 3.11-3.09 (m, 1H), 3.08-2.98 (m, 2H), 2.89 (d, J=12.0Hz, 1H), 2.20-2.00 (m, 3H), 1.89-1.78 (m, 1H), 0.97-0.95 (m, 4H).

Example 17 Synthesis of(2S,5R)-2-(5-((1-methylguanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 614)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1-methylguanidino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (614, 12 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):375.1. ¹H NMR (300 MHz, D₂O with coupe of drops of DMSO-d6) δ 6.55 (s,1H), 4.73 (s, 2H), 4.64 (d, J=6.0 Hz, 1H), 4.14 (br s, 1H), 3.15-3.07(m, 1H), 3.06 (s, 3H), 2.89 (d, J=12.2 Hz, 1H), 2.26-2.20 (m, 1H),2.15-2.09 (m, 2H), 1.92-1.85 (m, 1H).

Example 18 Synthesis of(2S,5R)-2-(5-(3-guanidinopropyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 615)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(3-guanidinopropyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (615, 18 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):389.1. ¹H NMR (300 MHz, D₂O with couple of drops of DMSO-d6) δ 6.28 (s,1H), 4.57 (d, J=6.5 Hz, 1H), 4.11 (s, 1H), 3.17 (t, J=6.7 Hz, 2H), 3.07(br d, J=11.5 Hz, 1H), 2.88 (d, J=12.4 Hz, 1H), 2.83 (t, J=6.7 Hz, 2H),2.22-1.75 (m, 6H).

Example 19 Synthesis of(2S,5R)-2-(5-((1s,3R)-3-aminocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 616)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1s,3R)-3-aminocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (616, 7 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):359.0. ¹H NMR (300 MHz, DMSO) δ 6.43 (s, 1H), 4.46 (d, J=6.1 Hz, 1H),4.02 (br s, 1H), 3.75-3.68 (m, 1H), 3.51-3.45 (m, 1H), 2.94-2.85 (m,1H), 2.72-2.60 (m, 3H), 2.36-2.30 (m, 2H), 2.15-1.95 (m, 1H), 1.99-1.90(m, 2H), 1.78-1.72 (m, 1H).

Example 20 Synthesis of(2S,5R)-2-(5-((1s,3R)-3-guanidinocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 617)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1s,3R)-3-guanidinocyclobutyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (617, 37 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):401.0. ¹H NMR (300 MHz, DMSO) δ 8.11 (br s, 1H), 7.14 (brs, 4H), 6.41(s, 1H), 4.45 (d, J=6.3 Hz, 1H), 4.02 (br s, 2H), 3.40-3.30 (m, 1H),2.91 (br d, J=10.3 Hz, 1H), 2.80-2.63 (m, 3H), 2.30-2.10 (m, 3H),2.00-1.85 (m, 2H), 1.82-1.66 (m, 1H).

Example 21 Synthesis of(2S,5R)-2-(5-(azetidin-3-ylmethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 618,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(azetidin-3-ylmethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (618, 75 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):359.0. ¹H NMR (300 MHz, D₂O) δ 6.23 (s, 1H), 4.56 (d, J=6.6 Hz, 1H),4.15-4.08 (m, 2H), 3.93-3.82 (m, 2H), 3.44 (dd, J=14.3, 7.2 Hz, 1H),3.28 (dt, J=15.9, 7.9 Hz, 1H), 3.10-3.03 (m, 3H), 2.87 (d, J=12.1 Hz,1H), 2.21-1.94 (m, 3H), 1.84-1.76 (m, 1H).

Example 22 Synthesis of(2S,5R)-2-(5-((1-carbamimidoylazetidin-3-yl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 619,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((1-carbamimidoylazetidin-3-yl)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (619, 45 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):401.0. ¹H NMR (300 MHz, DMSO) δ 7.28 (br s, 4H), 6.36 (s, 1H), 4.45 (d,J=6.8 Hz, 1H), 4.18 (t, J=8.4 Hz, 2H), 4.02 (br s, 1H), 3.86-3.71 (m,2H), 3.16 (d, J=7.0 Hz, 2H), 3.09-3.04 (m, 1H), 2.90 (br d, J=8.7 Hz,1H), 2.67 (d, J=11.8 Hz, 1H), 2.16-2.09 (m, 1H), 2.00-1.90 (m, 2H),1.80-1.72 (m, 1H).

Example 23 Synthesis of(2S,5R)-2-(5-(2-((2-aminoethyl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 620)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-((2-aminoethyl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (620, 20 mg, as formate salt) was obtained as a whitesolid after prep-HPLC purification using ammonium formate buffer. ESI-MS(EI⁺, m/z): 376.3. ¹H NMR (300 MHz, D₂O) δ 8.32 (s, 1H), 6.35 (s, 1H),4.59 (d, J=6.5 Hz, 1H), 4.11 (br s, 1H), 3.34 (t, J=6.8 Hz, 2H),3.30-3.13 (m, 6H), 3.11-3.03 (m, 1H), 2.91 (d, J=12.2 Hz, 1H), 2.21-1.98(m, 3H), 1.88-1.78 (m, 1H).

Example 24 Synthesis of(2S,5R)-2-(5-(2-((2-guanidinoethyl)amino)ethyl(isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 621,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-((2-guanidinoethyl)amino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (612, 15 mg, as formate salt) was obtained as a whitesolid after prep-HPLC purification using ammonium formate buffer. ESI-MS(EI⁺, m/z): 418.0. ¹H NMR (300 MHz, D₂O) δ 8.28 (s, 1H), 6.30 (s, 1H),4.55 (d, J=5.9 Hz, 1H), 4.07 (br s, 1H), 3.40 (t, J=5.9 Hz, 2H), 3.25(t, J=6.1 Hz, 2H), 3.14-3.01 (m, 5H), 2.87 (d, J=12.1 Hz, 1H), 2.16-2.09(m, 1H), 2.07-1.92 (m, 2H), 1.83-1.74 (m, 1H).

Example 25 Synthesis of(2S,5R)-2-(5-((azetidin-3-ylamino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 622,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((azetidin-3-ylamino)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (622, 267 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):374.0. ¹H NMR (400 MHz, D₂O) δ 6.55 (s, 1H), 4.66 (d, J=6.6 Hz, 1H),4.29-4.10 (m, 5H), 4.10-3.98 (m, 2H), 3.12 (d, J=12.0 Hz, 1H), 2.91 (d,J=12.1 Hz, 1H), 2.30-2.18 (m, 1H), 2.18-1.99 (m, 2H), 1.97-1.78 (m, 1H).

Example 26 Synthesis of(2S,5R)-2-(5-(2-(azetidin-3-ylamino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 623,)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-(azetidin-3-ylamino)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (623, 41 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):388.0.

Example 27 Synthesis of(2S,5R)-2-(5-((azetidin-3-yloxy)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 624)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-((azetidin-3-yloxy)methyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (624, 349 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):375.2. ¹H NMR (300 MHz, D₂O) δ 6.51 (s, 1H), 4.64 (s, 2H), 4.62 (d,J=7.2 Hz, 1H), 4.56-4.52 (m, 1H), 4.20 (dd, J=12.5, 6.7 Hz, 2H), 4.11(br s, 1H), 3.93 (dd, J=12.3, 5.2 Hz, 2H), 3.08 (br d, J=12.4 Hz, 1H),2.87 (d, J=12.2 Hz, 1H), 2.22-2.14 (m, 1H), 2.12-2.00 (m, 2H), 187-1.78(m, 1H).

Example 28 Synthesis of(2S,5R)-2-(5-(1-methylpyridin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylsulfate (Compound 625)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(1-methylpyridin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylsulfate (625, 20 mg) was obtained as a white solid. ESI-MS (EI⁻, m/z):379.0. ¹H NMR (400 MHz, d6-DMSO) δ 9.15 (d, J=5.2 Hz, 2H), 8.60 (d,J=5.2 Hz, 2H), 7.86 (s, 1H), 4.66 (d, J=5.6 Hz, 1H), 4.36 (s, 3H), 4.07(br s, 1H), 3.00-2.98 (m, 1H), 2.79 (d, J=12.2 Hz, 1H), 2.24-2.20 (m,1H), 2.10-2.00 (m, 2H), 1.82-1.78 (m, 1H).

Example 29 Synthesis of(2S,5R)-2-(5-(1,1-dimethylpiperidin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylsulfate (Compound 626)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(1,1-dimethylpiperidin-1-ium-4-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylsulfate (626, 86 mg) was obtained as a white solid after prep-HPLCpurification using ammonium formate buffer. ESI-MS (EI⁻, m/z): 399.2. ¹HNMR (400 MHz, CF₃COOD): δ 7.24 (s, 1H), 6.03 (d, J=6.8 Hz, 1H), 5.44 (brs, 1H), 4.76-4.72 (m, 1H), 4.51 (d, J=12.2 Hz, 1H), 4.26-4.12 (m, 4H),3.99-3.92 (m, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.26-3.22 (m, 1H),3.21-2.85 (m, 7H).

Example 30 Synthesis of(2S,5R)-7-oxo-2-(5-(piperidin-4-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 627)

Synthetic Scheme:

Characterization:

(2S,5R)-7-oxo-2-(5-(piperidin-4-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (627, 31 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):387.0.

Example 31 Synthesis of(2S,5R)-2-(5-(azetidin-3-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 628)

Characterization:

(2S,5R)-2-(5-(azetidin-3-yl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (628, 15 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):345.0. ¹H NMR (300 MHz, D₂O) δ 6.48 (s, 1H), 4.62 (d, J=5.2 Hz, 1H),4.47-4.22 (m, 5H), 4.15-4.12 (m, 1H), 3.17-3.03 (m, 1H), 2.91 (d, J=12.0Hz, 1H), 2.24-2.17 (m, 1H), 2.12-2.00 (m, 2H), 1.90-1.80 (m, 1H).CB-606, 122.

Example 32 Synthesis of(2S,5R)-2-(5-(2-(azetidin-3-yl)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 629)

Synthetic Scheme:

Characterization:

(2S,5R)-2-(5-(2-(azetidin-3-yl)ethyl)isoxazol-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (629, 34 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):373.0. ¹H NMR (300 MHz, DMSO) δ 8.33 (br s, 2H), 6.33 (s, 1H), 4.45 (d,J=6.4 Hz, 1H), 4.01 (br s, 1H), 3.92 (t, J=9.0 Hz, 2H), 3.61 (t, J=9.0Hz, 2H), 3.40-3.26 (m, 1H), 2.94-2.88 (m, 1H), 2.85-2.70 (m, 4H),2.16-2.08 (m, 1H), 2.06-1.87 (m, 3H), 1.84-1.67 (m, 1H).

Example 33 Synthesis of(2S,5R)-7-oxo-2-(5-(pyrrolidin-3-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (Compound 630)

Synthetic Scheme:

Characterization:

(2S,5R)-7-oxo-2-(5-(pyrrolidin-3-ylmethyl)isoxazol-3-yl)-1,6-diazabicyclo[3.2.1]octan-6-ylhydrogen sulfate (630, 9 mg) was obtained as a white solid afterprep-HPLC purification using ammonium formate buffer. ESI-MS (EI⁺, m/z):371.0. ¹H NMR (300 MHz, D₂O) δ 6.29 (s, 1H), 4.59 (d, J=6.6 Hz, 1H),4.12 (br s, 1H), 3.43 (dd, J=7.6, 5.0 Hz, 1H), 3.39-3.29 (m, 1H),3.26-3.14 (m, 1H), 3.12-3.03 (m, 1H), 2.97-2.85 (m, 4H), 2.74-2.61 (m,1H), 2.20-2.00 (m, 4H), 1.87-1.78 (m, 1H), 1.74-1.60 (m, 1H).

Example 34 Construction of Isogenic β-Lactamase Strains

A set of β-lactamase expressing isogenic E. coli strains was constructedby cloning a β-lactamase gene into a customized derivative of pBR322(GenBank Accession Number J01749) and transforming the engineeredplasmids into E. coli. The NdeI restriction site within the plasmidbackbone of pBR322 was removed to generate pBR322 ΔNdeI. The pBR322ΔNdeI vector itself, minus the blaTEM-1 gene, was amplified using twoprimers: (1) pBR-Pbla 5′-cgcatatgactettectttttcaatattattg-3, SEQ ID 1, aprimer with an engineered NdeI restriction site at the 3′ end of theblaTEM-1 promoter and (2) pBR-vec-1 5′-gcggatccctgtcagaccaagtttactc-3′,SEQ ID 2, a primer with an engineered BamHI restriction site at the 3′end of the blaTEM-1 open reading frame. The chloramphenicol resistancegene, cat, was generated by PCR amplification from pKD3 (GenBankAccession Number AY048742) using primers with an engineered NdeIrestriction site at the 5′ end (Pbla-cat5′-gccatatgatggagaaaaaaatcactgg-3′, SEQ ID 3) and an engineered BamHIrestriction site at the 3′ end (Vec-1-cat5′-cgggatccctagagaataggaacttcgg-3′, SEQ ID 4) of the resistance gene.The two PCR products, pBR322 ΔNdeI and cat were ligated togethergenerating pBR-CBST (pBR322 ΔNdeI ΔTEM-1::cat Seq. ID 5) which retainsboth the pBR322 tetracycline resistance cassette, tetA, and the plasmidorigin of replication but the blaTEM-1 gene was replaced by the catgene.

Using this engineering strategy a number of plasmids producingβ-lactamase genes from different classes (see below) were generatedusing synthetic genes with an engineered NdeI restriction site at the 5′end and BamHI restriction site at the 3′ end of each gene (GenScript).Both the synthetic β-lactamase genes and cat gene were ligated into theNdeI/BamHI sites of the pBR322 ΔNdeI PCR product and transformed intoelectrocompetent E. coli ElectroMax DH10B (Invitrogen/LifeTechnologies). E. coli DH10B harboring the recombinant plasmids wereselected on LB agar (supplemented with 25 μg/mL tetracycline) and singleisolated colonies were then inoculated into 5 mL LB media (supplementedwith 25 μg/mL tetracycline), and incubated at 37° C. with aeration (250rpm) for 18 hrs. The cultures were frozen back at −80° C. in 20%glycerol. The DNA sequence of the cloned β-lactamase genes wasconfirmed. The β-lactamase gene expression in the recombinant E. colistrains was driven by the blaTEM-1 promoter in the pBR-CBST plasmid andwas characterized by MIC profiling of the E. coli recombinant strainsagainst comparator β-lactam/BLI combinations in broth microdilutionassay.

GenBank Accession Number of β- β- Species β-Lacta- Lactamase Name & SEQ.ID of Lacta- Origin of mase Expressing plasmids producing maseβ-Lactamase Gene Strain β-Lactamase Class Gene Sequence KPC-2pBR-CBST-KPC-2 A K. pneumoniae EU784136 SEQID 6 CTX-M-15pBR-CBST-CTX-M-15 A K. pneumoniae JF775516 SEQ ID 7 SHV-12pBR-CBST-SHV-12 A K. pneumoniae AY008838 SEQ ID 8 P99 AmpC pBR-CBST-P99AMPC C E. cloacea XO7274 SEQ ID 9 OXA-15 pBR-CBST-OXA-15 D P. aeruginosaPAU63835 SEQ ID 10 KPC-4 pBR-CBST-KPC-4 A K. pneumoniae EU447304 SEQ ID11 DHA-1 pBR-CBST-DHA-1 C K. pneumoniae AY585202 SEQ ID 12 ADC-33pBR-CBST-ADC-33 C A. baumannii EU687478 SEQ ID 13Nucleotide Sequences of pBR-CBST Plasmids (Containing β-Lactamase or catGenes) Used in the E. coli Isogenic Strains (relevant restriction sitesare underlined; β-lactamase sequences in all caps, tetA sequence is initalics)

pBR-CBST-cat SEQ ID 5ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATACGGAATTTCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGTGGCAGGGCGGGGCGTAAGGCGCGCCATTTAAATGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaapBR-CBST-KPC-2 SEQ ID 6ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGTCACTGTATCGCCGTCTAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGCCACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGACTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAAATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACAACAGGCATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGTGATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGGCCGGGCTGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCCGTCTGGACCGCTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACAAAAACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGACTGGGCAGTCGGAGACAAAACCGGAACCTGCGGAGTGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGGCGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCGGCTAGACTCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaapBR-CBST-CTX-M-15 SEQ ID 7ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTCCGGGCGATCCGCGTGATACCACTTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGGGCGCAGCTGGTGACATGGATGAAAGGCAATACCACCGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGCAGCGGTGGCTATGGCACCACCAACGATATCGCGGTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCACTTACTTCACCCAGCCTCAACCTAAGGCAGAAAGCCGTCGCGATGTATTAGCGTCGGCGGCTAAAATCGTCACCGACGGTTTGTAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctgaacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgcccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-SHV-12 SEQ ID 8ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGCGTTATATTCGCCTGTGTATTATCTCCCTGTTAGCCACCCTGCCGCTCGCGGTACACGCCAGCCCGCAGCCGCTTGAGCAAATTAAACAAAGCGAAAGCCAGCTGTCGGGCCGCGTAGGCATGATAGAAATGGATCTGGCCAGCGGCCGCACGCTGACCGCCTGGCGCGCCGATGAACGCTTTCCCATGATGAGCACCTTTAAAGTAGTGCTCTGCGGCGCAGTGCTGGCGCGGGTGGATGCCGGTGACGAACAGCTGGAGCGAAAGATCCACTATCGCCAGCAGGATCTGGTGGACTACTCGCCGGTCAGCGAAAAACACCTTGCCGACGGCATGACGGTCGGCGAACTCTGCGCCGCCGCCATTACCATGAGCGATAACAGCGCCGCCAATCTGCTGCTGGCCACCGTCGGCGGCCCCGCAGGATTGACTGCCTTTTTGCGCCAGATCGGCGACAACGTCACCCGCCTTGACCGCTGGGAAACGGAACTGAATGAGGCGCTTCCCGGCGACGCCCGCGACACCACTACCCCGGCCAGCATGGCCGCGACCCTGCGCAAGCTGCTGACCAGCCAGCGTCTGAGCGCCCGTTCGCAACGGCAGCTGCTGCAGTGGATGGTGGACGATCGGGTCGCCGGACCGTTGATCCGCTCCGTGCTGCCGGCGGGCTGGTTTATCGCCGATAAGACCGGAGCTAGCAAGCGGGGTGCGCGCGGGATTGTCGCCCTGCTTGGCCCGAATAACAAAGCAGAGCGCATTGTGGTGATTTATCTGCGGGATACCCCGGCGAGCATGGCCGAGCGAAATCAGCAAATCGCCGGGATCGGCGCGGCGCTGATCGAGCACTGGCAACGCTAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggacccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtgaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-P99 SEQ ID 9ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGATGAGAAAATCCCTTTGCTGCGCCCTGCTGCTCGGCATCTCTTGCTCTGCTCTCGCCACGCCAGTGTCAGAAAAACAGCTGGCGGAGGTGGTCGCGAATACGATTACCCCGCTGATGAAAGCCCAGTCTGTTCCAGGCATGGCGGTGGCCGTTATTTATCAGGGAAAACCGCACTATTACACATTTGGCAAGGCCGATATCGCGGCGAATAAACCCGTTACGCCTCAGACCCTGTTCGAGCTGGGTTCTATAAGTAAAACCTTCACCGGCGTTTTAGGTGGGGATGCCATTGCTCGCGGTGAAATTTCGCTGGACGATGCGGTGACCAGATACTGGCCACAGCTGACGGGCAAGCAGTGGCAGGGTATTCGTATGCTGGATCTCGCCACCTACACCGCTGGCGGCCTGCCGCTACAGGTACCGGATGAGGTCACGGATAACGCCTCCCTGCTGCGCTTTTATCAAAACTGGCAGCCGCAGTGGAAGCCTGGCACAACGCGTCTTTACGCCAACGCCAGCATCGGTCTTTTTGGTGCGCTGGCGGTCAAACCTTCTGGCATGCCCTATGAGCAGGCCATGACGACGCGGGTCCTTAAGCCGCTCAAGCTGGACCATACCTGGATTAACGTGCCGAAAGCGGAAGAGGCGCATTACGCCTGGGGCTATCGTGACGGTAAAGCGGTGCGCGTTTCGCCGGGTATGCTGGATGCACAAGCCTATGGCGTGAAAACCAACGTGCAGGATATGGCGAACTGGGTCATGGCAAACATGGCGCCGGAGAACGTTGCTGATGCCTCACTTAAGCAGGGCATCGCGCTGGCGCAGTCGCGCTACTGGCGTATCGGGTCAATGTATCAGGGTCTGGGCTGGGAGATGCTCAACTGGCCCGTGGAGGCCAACACGGTGGTCGAGGGCAGCGACAGTAAGGTAGCACTGGCGCCGTTGCCCGTGGCAGAAGTGAATCCACCGGCTCCCCCGGTCAAAGCGTCCTGGGTCCATAAAACGGGCTCTACTGGCGGGTTTGGCAGCTACGTGGCCTTTATTCCTGAAAAGCAGATCGGTATTGTGATGCTCGCGAATACAAGCTATCCGAACCCGGCACGCGTTGAGGCGGCATACCATATCCTCGAGGCGCTACAGTAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaapBR-CBST-OXA-15 SEQ ID 10ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGGCAATCCGAATCTTCGCGATACTTTTCTCCATTTTTTCTCTTGCCACTTTCGCGCATGCGCAAGAAGGCACGCTAGAACGTTCTGACTGGAGGAAGTTTTTCAGCGAATTTCAAGCCAAAGGCACGATAGTTGTGGCAGACGAACGCCAAGCGGATCGTGCCATGTTGGTTTTTGATCCTGTGCGATCGAAGAAACGCTACTCGCCTGCATCGACATTCAAGATACCTCATACACTTTTTGCACTTGATGCAGGCGCTGTTCGTGATGAGTTCCAGATTTTTCGATGGGACGGCGTTAACAGGGGCTTTGCAGGCCACAATCAAGACCAAGATTTGCGATCAGCAATGCGGAATTCTACTGTTTGGGTGTATGAGCTATTTGCAAAGGAAATTGGTGATGACAAAGCTCGGCGCTATTTGAAGAAAATCGACTATGGCAACGCCGGTCCTTCGACAAGTAATGGCGATTACTGGATAGAAGGCAGCCTTGCAATCTCGGCGCAGGAGCAAATTGCATTTCTCAGGAAGCTCTATCGTAACGAGCTGCCCTTTCGGGTAGAACATCAGCGCTTGGTCAAGGATCTCATGATTGTGGAAGCCGGTCGCAACTGGATACTGCGTGCAAAGACGGGCTGGGAAGGCCGTATGGGTTGGTGGGTAGGATGGGTTGAGTGGCCGACTGGCTCCGTATTCTTCGCACTGAATATTGATACGCCAAACAGAATGGATGATCTTTTCAAGAGGGAGGCAATCGTGCGGGCAATCCTTCGCTCTATTGAAGCGTTACCGCCCAACCCGGCAGTCAACTCGGACGCTGCGCGATAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa pBR-CBST-KPC-4SEQ ID 11ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGTCACTGTATCGCCGTCTAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGCCACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGACTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAAATGCGCTGGTTCGGTGGTCACCCATCTCGGAAAAATATCTGACAACAGGCATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGTGATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGGCCGGGCTGACGGCCTTCATGCGCTCTATCGGCGATACCACGTTCCGTCTGGACCGCTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACAAAAACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGACTGGGCAGTCGGAGACAAAACCGGAACCTGCGGAGGGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGGCGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCGGCTAGACTCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccaattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatgggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaapBR-CBST-DHA-1 SEQ ID 12ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGAAAAAATCGTTATCTGCAACACTGATTTCCGCTCTGCTGGCGTTTTCCGCCCCGGGGTTTTCTGCCGCTGATAATGTCGCGGCGGTGGTGGACAGCACCATTAAACCGCTGATGGCACAGCAGGATATTCCCGGGATGGCGGTTGCCGTCTCCGTAAAGGGTAAGCCCTATTATTTCAATTATGGTTTTGCCGATATTCAGGCAAAACAGCCGGTCACTGAAAATACACTATTTGAGCTCGGATCTGTAAGTAAAACTTTCACAGGTGTGCTGGGTGCGGTTTCTGTGGCGAAAAAAGAGATGGCGCTGAATGATCCGGCGGCAAAATACCAGCCGGAGCTGGCTCTGCCGCAGTGGAAGGGGATCACATTGCTGGATCTGGCTACCTATACCGCAGGCGGACTGCCGTTACAGGTGCCGGATGCGGTAAAAAGCCGTGCGGATCTGCTGAATTTCTATCAGCAGTGGCAGCCGTCCCGGAAACCGGGCGATATGCGTCTGTATGCAAACAGCAGTATCGGCCTGTTTGGTGCTCTGACCGCAAACGCGGCGGGGATGCCGTATGAGCAGTTGCTGACTGCACGCATCCTGGCACCGCTGGGGTTATCTCACACCTTTATTACTGTGCCGGAAAGTGCGCAAAGCCAGTATGCGTACGGTTATAAAAACAAAAAACCGGTCCGCGTGTCGCCGGGACAGCTTGATGCGGAATCTTACGGCGTGAAATCCGCCTCAAAAGATATGCTGCGCTGGGCGGAAATGAATATGGAGCCGTCACGGGCCGGTAATGCGGATCTGGAAATGGCAATGTATCTCGCCCAGACCCGCTACTATAAAACCGCCGCGATTAACCAGGGGCTGGGCTGGGAAATGTATGACTGGCCGCAGCAGAAAGATATGATCATTAACGGTGTGACCAACGAGGTCGCATTGCAGCCGCATCCGGTAACAGACAACCAGGTTCAGCCGTATAACCGTGCTTCCTGGGTGCATAAAACGGGCGCAACAACTGGTTTCGGCGCCTATGTCGCCTTTATTCCGGAAAAACAGGTGGCGATTGTGATTCTGGCGAATAAAAACTACCCGAATACCGAAAGAGTCAAAGCTGCACAGGCTATTTTGAGTGCACTGGAATAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcg agtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggcacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaapBR-CBST-ADC-33 SEQ ID 13ttcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcatATGCGATTTAAAAAAATTTCTTGTCTACTTTTATCCCCGCTTTTTATTTTTAGTACCTCAATTTATGCGGGCAATACACCAAAAGACCAAGAAATTAAAAAACTGGTAGATCAAAACTTTAAACCGTTATTAGAAAAATATGATGTGCCAGGTATGGCTGTGGGTGTTATTCAAAATAATAAAAAGTATGAAATGTATTATGGTCTTCAATCTGTTCAAGATAAAAAAGCCGTAAATAGCAGTACCATTTTTGAGCTAGGTTCTGTCAGTAAATTATTTACCGCGGACAGCAGGTGGATATGCAAAAAATAAAGGAAAAATCTCTTTTGACGATACGCCTGGTAAATATTGGAAAGAACTAAAAAACACACCGAITGACCAAGTTAACTTACTTCAACTCGCGACGTATACAAGTGGTAACCTTGCCTTGCAGTTTCCAGATGAAGTAAAAACAGACCAACAAGTTTTAACTTTTTTCAAAGACTGGAAACCTAAAAACTCAATCGGTGAATACAGACAATATTCAAATCCAAGTATTGGCCTATTTGGAAAGGTTGTGGCTTTGTCTATGAATAAACCTTTCGACCAAGTCTTAGAAAAAACAATTTTTCCGGCCCTTGGCTTAAAACATAGCTATGTAAATGTACCTAAGACCCAGATGCAAAACTATGCATTTGGTTATAACCAAGAAAATCAGCCGATTCGAGTTAACCGCGGCCCACTCGATGCCGCCCCTGCGTATGGCGTCAAATCGACACTACCCGACATGTTGAGTTTTATTCATGCCAACCTTAACCCACAGAAATATCCGGCTGATATTCAACGGGCAATTAATGAAACACATCAAGGGCGCTATCAAGTAAATACCATGTATCAGGCACTCGGTTGGGAAGAGTTTTCTTATCCGGCAACGTTACAAACTTTATTAGACAGTAATTCAGAACAGATTGTGATGAAACCTAATAAAGTGACTGCTATTTCAAAGGAACCTTCAGTTAAGATGTACCATAAAACTGGCTCAACCAACGGTTTCGGAACGTATGTAGTGTTTATTCCTAAAGAAAATATTGGCTTAGTCATGTTAACCAATAAACGTATTCCAAATGAAGAGCGCATTAAGGCAGCTTATGCTGTGCTGAATGCAATAAAGAAATAAggatccctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttctgcgcgtaatctgctgcttgcaaacaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatttggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccagtgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgccagcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggattcacaggacgggtgtggtcgccatgatcgcgtagtcgatagtggctccaagtagcgaagcgagcaggactgggcggcggccaaagcggtcggacagtgctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagcagcacgccatagtgactggcgatgctgtcggaatggacgatatcccgcaagaggcccggcagtaccggcataaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattaaagcttatcgatgataagctgtcaaacatgagaa

Example 35 Standard BLI Potentiation MIC Assay

The ability of compounds to potentiate the activity of β-lactams wasdemonstrated by determining the minimum inhibitory concentrations (MIC)of β-lactam and BLI compound combinations against various β-lactamaseproducing bacterial strains using the broth microdilution method. Theexperimental protocol was performed according to Clinical and LaboratoryStandards Institute (CLSI) guidelines with modifications as describedbelow (CLSI guidelines can be derived from the CLSI document M07-A9published in January 2012: “Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria That Grow Aerobically; ApprovedStandard-Ninth Edition”).

To prepare for MIC testing, frozen glycerol stocks of clinical isolates(Klebsiella pneumoniae, Eschericia coli, Enterobacter spp, Citrobacterspp, or Pseudomonas aeruginosa) were used to streak for isolatedcolonies on rich, non-selective, tryptic soy agar containing 5% sheep'sblood (TSAB). Frozen glycerol stocks of laboratory engineered, isogenicE. coli strains, which contain cloned β-lactamase expressing plasmidswere used to streak for isolated colonies on rich, selective LB agarsupplemented with 25 μg/mL tetracycline to maintain the plasmid. Allstrains were incubated at 37° C. for 18-24 hrs.

On the day of testing, primary cultures were started by scraping off5-10 colonies from the TSAB plates containing clinical strains or thetetracycline supplemented LB plates containing engineered strains. Theclinical strain material was suspended in ˜5 mL of cation adjustedMueller Hinton Broth (CAMHB) in 14 mL culture tubes. The engineeredstrain material was suspended in CAMHB (supplemented with 25 μg/mLtetracycline) in 14 mL culture tubes. All strains were incubated at 37°C. with aeration (200 rpm) for ˜2 hrs until the optical density at 600nm (OD600) was ≧0.1.

The two compound components of the assay were each diluted in CAMHB andadded to the 96-well broth microdilution assay plates. 50 μL of theβ-lactam was added to each well of the assay plate in 2-fold dilutionswith final concentrations ranging from 128 to 0.13 μg/mL. 25 μL of theBLI compound was added to all wells in the broth microdilution plates ata final concentration of 4 μg/mL. Inoculum cultures were prepared bystandardizing the primary cultures to OD600=0.1 and then adding 20 μL ofthe adjusted primary culture per 1 mL CAMHB for clinical strains orCAMHB (supplemented with tetracycline at 100 μg/mL) for engineeredstrains, so that the final inoculum density was ˜10⁵ colony formingunits per milliliter. Diluted inoculum cultures were used to inoculate25 μL per well in 96-well broth microdilution assay plates. The finalvolume of each well was 100 μL and contained a β-lactam at differentconcentrations, a BLI compound at 4 μg/mL concentration, the bacterialculture at an OD600 of approximately 0.001 and when necessarytetracycline at 25 μg/mL.

Plates were incubated for 18-20 hours at 37° C. with aeration (200 rpm).Following incubation, growth was confirmed visually placing plates overa viewing apparatus (stand with a mirror underneath) and then OD600 wasmeasured using a SpectraMax 340PC384 plate reader (Molecular Devices,Sunnyvale, Calif.). Growth was defined as turbidity that could bedetected with the naked eye or achieving minimum OD600 of 0.1. MICvalues were defined as the lowest concentration producing no visibleturbidity.

MIC values of representative compounds are shown in Table II.

Example 36 Synergy MIC (sMIC) Assay

The synergy MIC (sMIC) assay determines the concentration of the BLIrequired to potentiate the activity of a fixed concentration of aβ-lactam antibiotic against β-lactamase producing bacterial strains. Theexperimental protocol was performed according to Clinical and LaboratoryStandards Institute (CLSI) guidelines with modifications as describedbelow (CLSI guidelines can be derived from the CLSI document M07-A9published in January 2012: “Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria That Grow Aerobically; ApprovedStandard-Ninth Edition”). The assay is set-up by serially diluting theBLI across 11 of the 12 wells in each row of a 96-well brothmicrodilution assay plate, adding the β-lactam at a fixed concentrationto all wells in the assay plate, inoculating the assay plate withbacterial strains, and determining the lowest concentration of BLIrequired to inhibit overnight bacterial growth. Bacterial growth in the12^(th) well of the assay plate, which contains the β-lactam at a fixedconcentration but does not contain any BLI, demonstrates that thebacterial strains are resistant to the β-lactam antibiotic (e.gceftolozane) at the fixed concentration of 4 μg/mL.

To prepare for MIC testing, frozen glycerol stocks of clinical isolates(Klebsiella pneumoniae, Eschericia coli, Enterobacter spp, Citrobacterspp, or Pseudomonas aeruginosa) were used to streak for isolatedcolonies on rich, non-selective, tryptic soy agar containing 5% sheep'sblood (TSAB). Frozen glycerol stocks of laboratory engineered, isogenicE. coli strains, which contain cloned β-lactamase expressing plasmidswere used to streak for isolated colonies on rich, selective LB agarsupplemented with 25 μg/mL tetracycline to maintain the plasmid. Allstrains were incubated at 37° C. for 18-24 hrs.

On the day of testing, primary cultures were started by scraping off5-10 colonies from the TSAB plates containing clinical strains or thetetracycline supplemented LB plates containing engineered strains. Theclinical strain material was suspended in ˜5 mL of cation adjustedMueller Hinton Broth (CAMHB) in 14 mL culture tubes. The engineeredstrain material was suspended in CAMHB (supplemented with tetracyclineat 25 μg/mL) in 14 mL culture tubes. All strains were incubated at 37°C. with aeration (200 rpm) for ˜2 hrs until the OD600 was ≧0.1.

The two compound components of the assay were each prepared in CAMHB andadded to the 96-well broth microdilution assay plates. 50 μL of the BLIwas added to each well of the assay plate in 2-fold dilutions with finalconcentrations ranging from 128 to 0.13 μg/mL. 25 μL of the β-lactam wasadded to all wells in the broth microdilution plates at a finalconcentration of 4 μg/mL. Inoculum cultures were prepared bystandardizing the primary cultures to OD600=0.1 and then adding 20 μL ofthe adjusted primary culture per 1 mL CAMHB for clinical strains orCAMHB (supplemented with tetracycline at 100 μg/mL) for isogenicstrains, so that the final inoculum density was ˜10⁵ colony formingunits per milliliter. Diluted inoculum cultures were used to inoculate25 μL per well in 96-well broth microdilution assay plates. The finalvolume of each well was 100 μL and contained a BLI at differentconcentrations, a β-lactam at 4 μg/mL concentration, the bacterialculture at an OD600 of approximately 0.001 and when necessarytetracycline at 25 ug/mL.

Interpreting the sMIC Data:

Plates were incubated for 18-20 hours at 37° C. with aeration (200 rpm).Following incubation, growth was confirmed visually placing plates overa viewing apparatus (stand with a mirror underneath) and then OD600 wasmeasured using a SpectraMax 340PC384 plate reader (Molecular Devices,Sunnyvale, Calif.). Growth was defined as turbidity that could bedetected with the naked eye or achieving minimum OD600 of 0.1. sMICvalues were defined as the lowest concentration producing no visibleturbidity.

The sMIC values represent the amount of BLI required to potentiate theactivity of 4 μg/ml of CXA-101 (Ceftolozane) or ceftazidime to inhibitthe growth of the β-lactamase producing bacteria.

sMIC values of representative compounds are shown in Table III.

Example 37 Inhibition Kinetics

Inhibition or inactivation of KPC-2 by test inhibitors was assessedusing 100 μM nitrocefin (NCF) as a reporter substrate. Assays wereperformed in 1× PBS pH 7.4, 0.1 mg/ml BSA, in 96-well half area plates,50 μl reaction volume. NCF was dissolved in DMSO and diluted in assaybuffer. Test inhibitors were dissolved in water or DMSO and seriallydiluted in the assay with final concentrations between 2000-0.195 μM.The enzyme activity in the presence of varying concentrations of testinhibitor was determined by monitoring the hydrolysis of NCFspectrophotometrically at 486 nm, for 5 minutes, 25° C., using aSpectraMax Plus384 microplate reader with SoftMax Pro software(Molecular Devices). Data analysis was performed using GraphPad Prism(GraphPad Software, Inc.).

Progress curves were fit to a first-order rate decay equation (Eq. 1) todetermine k_(observed) (k_(obs)).k_(obs) vs. inhibitor concentration [I] curves were then fit to Eq. 2 todetermine the inhibitor dissociation constant (K) and the first orderrate constant of enzyme inactivation at infinite inhibitor concentration(k_(inact)). Table IV shows kinetics results from representative testcompounds. A larger k_(inact)/K ratio indicates a more effective enzymeinactivator.

Y _(t) =V ₀*(1−e ^((−k) ^(obs) ^(*t)))/k _(obs)  Eq. 1

Where Y is the absorbance at time t, V₀ is the uninhibited enzymevelocity, k_(obs) is the observed rate constant of the enzymeinactivation.

k _(obs) =k _(inact) *[I]/([I]+K(1+S/K _(m)))  Eq. 2

Where S is the NCF concentration, K_(m) is the KPC-2 K_(m) for NCF

1-50. (canceled)
 51. A pharmaceutical composition comprising ceftolozaneor a pharmaceutically acceptable salt thereof and a compound of Formula(A-I) or a pharmaceutically acceptable salt thereof:

wherein R* is selected from

and R^(1*) is selected from: a.

wherein R^(2*) is selected from

R^(3*) is selected from hydrogen, (C₁-C₃)-alkyl, aminoalkyl,aminocycloalkyl, hydroxyalkyl,

each of R^(4*), R^(5*), R^(6*)and R^(7*) is independently selected fromhydrogen, (C₁-C₆)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl,provided that at least one of R^(4*), R^(5*), R^(6*)and R^(7*)ishydrogen, n is selected from 1, 2, 3 and 4, and m is selected from 1, 2and 3; b.

wherein R⁸ is selected from —NH(C₁-C₃)-alkyl and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously; c.

wherein Z is selected from CR⁹R¹⁰ and NR¹¹, each of R⁹ and R¹⁰ isindependently selected from H, NH₂, —NH(C₁-C₃)-alkyl and

wherein each of R^(4*), R^(5*), R⁶ and R^(7*)is as described previously,alternatively, R⁹ and R¹⁰ together with the carbon to which they areattached, form a cycloalkyl or heterocyclyl ring containing 4-6 ringmembers, R¹¹ is selected from H and

each of R¹², R¹³ and R¹⁴ is independently selected from hydrogen,(C₁-C₆)-alkyl, amino alkyl, aminocycloalkyl, and hydroxyalkyl, providedthat at least one of R¹², R¹³ and R¹⁴ is hydrogen, R¹⁵ is selected fromNH₂ and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously, each of p* and q* is independently selected from 0, 1, 2 and3, T is selected from NH and O, t is selected from 0, 1, 2, 3, and 4,and each of r and y is independently selected from 0 and 1; d.

wherein R¹⁶ is selected from NH₂, —NH(C₁-C₃)-alkyl and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*) is as describedpreviously, s is selected from 0 and 1, and, v is selected from 0, 1, 2,and 3; e.

wherein R¹⁸ is selected from NH₂ and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*) is as describedpreviously, R¹⁷ is selected from amino and hydroxyl, and w is selectedfrom 0 and 1; f.

wherein M is selected from NR¹⁹, CR²⁰R²¹ and O, wherein R¹⁹ is selectedfrom H and

where each of R¹², R¹³ and R¹⁴ is as described previously, each of R²⁰and R²¹ is independently selected from H, NH₂ and

wherein each of R^(4*), R^(5*), R^(6*)and R^(7*)is as describedpreviously, and u is selected from 0, 1 and 2; and g.


52. The pharmaceutical composition according to claim 51, wherein thecompound of Formula A-I has the stereochemistry specified in FormulaA-II

or a pharmaceutically acceptable salt thereof.
 53. The pharmaceuticalcomposition according to claim 52, wherein the compound, or apharmaceutically acceptable salt thereof is selected from


54. The pharmaceutical composition according to claim 51, wherein R^(1*)is selected from


55. The pharmaceutical composition according to claim 53, wherein thecompound is selected from


56. The pharmaceutical composition according to claim 55, wherein thecompound or a pharmaceutically acceptable salt thereof has the Formula


57. The pharmaceutical composition compound according to claim 55,wherein the compound or a pharmaceutically acceptable salt thereof hasthe Formula